We have examined the distribution of the endothelial isoform of nitric oxide synthase (eNOS) in villous and extravillous trophoblast populations by immunohistochemistry and have further studied expression of eNOS during differentiation of cytotrophoblast into syncytiotrophoblast in culture. In first trimester villous tissue, NADPH diaphorase activity and eNOS immunostaining were present in syncytiotrophoblast but not the progenitor cytotrophoblast layer. Extravillous trophoblast in the basal plate of the placenta was identified by anticytokeratin immunostaining and displayed NADPH diaphorase activity, but not eNOS immunostaining. Both amnion epithelial cells and chorion cytotrophoblast had NADPH diaphorase activity but no eNOS immunostaining, whereas eNOS immunostaining was seen in the fibroblast layer of amnion. Purified villous cytotrophoblast cells from term placentae aggregated and fused to form a syncytium with increasing time in culture as assessed by antidesmosomal protein and antinuclear antibody immunostaining. Following 24 h in culture, the majority of cells were still mononucleate cytotrophoblast which did not display eNOS immunostaining, whereas a few syncytial aggregates had formed which were both eNOS positive and hPL positive. By 3 to 5 days in culture, the majority of cells were present as syncytiotrophoblast. However, eNOS and hPL immunostaining was more diffuse and not all syncytial aggregates were positive. Of the trophoblast populations, only syncytiotrophoblast appears to express eNOS. Differentiation of cytotrophoblast into syncytiotrophoblast is associated with eNOS expression. © 1995.