Key points Muscle contraction is triggered by Ca2+ ions released from the sarcoplasmic reticulum (SR) in response to depolarization of skeletal muscle fibres. Muscle activation is also known to be associated with a voltage‐activated trans‐sarcolemmal Ca2+ influx. Because removal of external Ca2+ does not impede fibres from contracting, a negligible role has been initially attributed to this Ca2+ entry. Furthermore, it is not clearly established whether Ca2+ exclusively flows through L‐type channels. By monitoring the quenching of fura‐2 fluorescence resulting from Mn2+ influx in voltage‐controlled mouse and zebrafish muscle fibres, we show that the L‐type current is the only contributor to Ca2+ influx during long‐lasting depolarizations. Calibration of the Mn2+ quenching signal allowed us to estimate an Mn2+ current of 0.31 A F–1 flowing during a train of action potentials. Measurements of SR Ca2+ changes with fluo‐5N in response to depolarization indicated that voltage‐activated Ca2+/Mn2+ influx contributes to SR Ca2+/Mn2+ loading. Abstract Muscle contraction is triggered by Ca2+ ions released from the sarcoplasmic reticulum (SR) in response to depolarization of skeletal muscle fibres. Muscle activation is also associated with a voltage‐activated trans‐sarcolemmal Ca2+ influx early identified as a current flowing through L‐type Ca2+ channels. Because removal of external Ca2+ does not impede fibres from contracting, a negligible role was given to this voltage‐activated Ca2+ entry, although the decline of Ca2+ release is more pronounced in the absence of Ca2+ during long‐lasting activation. Furthermore, it is not clearly established whether Ca2+ exclusively flows through L‐type channels or in addition through a parallel voltage‐activated pathway distinct from L‐type channels. Here, by monitoring the quenching of fura‐2 fluorescence resulting from Mn2+ influx in voltage‐controlled mouse and zebrafish isolated muscle fibres, we show that the L‐type current is the only contributor to Ca2+ influx during long‐lasting depolarizations in skeletal muscle. Calibration of the Mn2+ quenching signal allowed us to estimate a mean Mn2+ current of 0.31 ± 0.06 A F–1 flowing through L‐type channels during a train of action potentials. Measurements of SR Ca2+ changes with fluo‐5N in response to depolarization revealed that an elevated voltage‐activated Ca2+ current potentiated SR Ca2+ loading and addition of external Mn2+ produced quenching of fluo‐5N in the SR, indicating that voltage‐activated Ca2+/Mn2+ influx contributes to SR Ca2+/Mn2+ loading.