Complex regulatory processes alter the activity of endothelial nitric oxide synthase (eNOS) leading to nitric oxide (NO) production by endothelial cells under various physiological states. These complex processes require specific subcellular eNOS partitioning between plasma membrane caveolar domains and non‐caveolar compartments. Translocation of eNOS from the plasma membrane to intracellular compartments is important for eNOS activation and subsequent NO biosynthesis. We present data reviewing and interpreting information regarding: (i) the coupling of endothelial plasma membrane receptor systems in the caveolar structure relative to eNOS trafficking; (ii) how eNOS trafficking relates to specific protein–protein interactions for inactivation and activation of eNOS; and (iii) how these complex mechanisms confer specific subcellular location relative to eNOS multisite phosphorylation and signalling. Dysfunction in the regulation of eNOS activation may contribute to several disease states, in particular gestational endothelial abnormalities (pre‐eclampsia, gestational diabetes etc.), that have life‐long deleterious health consequences that predispose the offspring to develop hypertensive disease, Type 2 diabetes and adiposity.