["The Journal of Physiology, Volume 604, Issue 7, Page 3032-3052, 1 April 2026. ", "\nAbstract figure legend In cortical layer (L)5 pyramidal neurons we found that mitochondria in the axon initial segment (AIS) are distributed non‐uniformly and display robust calcium (Ca2+) uptake during action potential (AP) firing. Because the AIS is the site for the initiation of APs, which are shaped by cytosolic Ca2+ levels, we hypothesized that mitochondrial Ca2+ buffering at the AIS could impact the AP waveform. However, when we pharmacologically blocked mitochondrial Ca2+ uptake, we only detected an increase in the duration of the afterhyperpolarization (AHP), while the AP initiation and waveforms were unaltered. These data suggest that mitochondria at the AIS play other, non‐electrical, roles.\n\n\n\n\n\n\n\n\n\nAbstract\nMitochondria are diverse and multifaceted intracellular organelles regulating oxidative energy supply, lipid metabolism and calcium (Ca2+) signalling. In neurons the spatial sequestration of cytoplasmic Ca2+ by mitochondria plays a critical role in determining activity‐dependent spine plasticity, shaping the presynaptic transmitter release characteristics and contributing to sustained action potential firing. Here, we tested the hypothesis that mitochondria at the axon initial segment (AIS) affect the microdomain cytoplasmic Ca2+ transients, thereby regulating Ca2+‐dependent voltage‐gated ion channels at the plasma membrane and initiation of action potentials. Using 3D electron microscopy reconstructions and virally injecting genetically encoded fluorescence indicators we visualized the ultrastructure and distribution of mitochondria selectively in thick‐tufted layer 5 pyramidal neurons. We found that most mitochondria were stably clustered to the proximal AIS, while few were observed at distal sites. Simultaneous two‐photon imaging of action potential‐dependent cytoplasmic and mitochondrial Ca2+, combined with electrophysiological recordings showed that AIS mitochondria exhibit powerful activity‐dependent cytosolic Ca2+ uptake. However, while intracellular application of the mitochondrial Ca2+ uniporter inhibitor Ru360 fully blocked mitochondrial Ca2+ import and increased the slow afterhyperpolarization duration, it did not affect action potential input–output function, action potential dynamics nor the ability to produce high‐frequency burst output. Together, the results indicate that AIS mitochondria are dispensable for temporal and rate encoding, suggesting that mt‐Ca2+ buffering at the AIS may be involved in non‐electrical roles.\n\n\n\n\n\n\n\n\n\nKey points\n\nMitochondrial Ca2+ buffering controls multiple Ca2+‐dependent intracellular processes and their subcellular location of the organelles defines local physiological properties in neurons.\nRecent studies implicate mitochondrial Ca2+ uptake in the slow afterhyperpolarization and maintenance of action potential firing.\nUsing electron microscopy and virally delivered genetically encoded tools we examined mitochondria in the layer 5 pyramidal neuron axon initial segment (AIS), the site where action potentials initiate, and found that cytoplasmic Ca2+ influx is powerfully buffered by proximally clustered mitochondria.\nElectrophysiological recordings during the block of the mitochondrial calcium uniporter reveal a role in the slow afterhyperpolarization, while AIS action potential initiation and action potential waveforms are independent from mitochondria.\nThese findings indicate AIS mitochondria under physiological conditions exert non‐electrical roles.\n\n\n"]