Endothelial nitric oxide synthase (eNOS) is expressed in the endothelium of placental vessels and also surprisingly in the syncytiotrophoblast lining of these vessels in the human placenta. eNOS is thought to play an important role in basal nitric oxide release to maintain maximal vasodilatation of fetal capillaries. Caveolin-1 is known to bind eNOS and inhibit it by compartmentalisation within caveolae. The aim of this study was to investigate the effects of L-NAME, the non-selective competitive inhibitor of eNOS, on the spatial localization of eNOS and caveolin-1 in the human placental vascular tree. Using a well-established dual perfusion system, placental lobules of normal term placentae (n=6) were perfused for 30 min with/without 5 mM L-NAME added to the fetal circulation, with continual monitoring of fetal perfusion pressure. Following perfusion fixation, tissue biopsies were frozen, cryosectioned and immunostained for eNOS and caveolin-1. Chorionic villi and vascular profiles within were visualized by fluorescence microscopy and quantitative analysis (blinded) of villous/vascular profiles was performed. In control perfused placenta eNOS was located predominantly in the endothelium of larger stem and intermediate villous vessels at the expense of terminal villous capillaries. A similar differential expression was seen for caveolin-1. Both eNOS and Cavelolin-1 was also localised to the syncytiotrophoblast. In L-NAME perfused placentae there was an altered pattern of eNOS staining in the syncytiotrophoblast: from a mixed punctate (caveolar/vesicular) and diffuse (cytosolic) localization to a predominantly punctate staining pattern (p≤0.05). An increase in caveolin -1 positive puncta (frequency and intensity) was also seen in the L-NAME group. The induced alteration in expression profiles of both proteins suggest that L-NAME inhibits release of eNOS from the caveolae in the syncytiotrophoblast and this also allows a greater visualisation of caveolin-1. This study also highlights the role of the syncytiotrophoblast in regulating vascular tone of fetal capillaries in the human placenta.