Experiments were designed to determine whether or not aging per se or cellular density affects endothelial NO-synthase (eNOS) activity in cultured coronary endothelial cells of the pig. A diminished activity could explain the reduced endothelium-dependent relaxation to bradykinin previously observed during regeneration after endothelial injury. The results demonstrate that cell cultures derived from eight-day old regenerated endothelium exhibit a normal basal production of cyclic GMP, but a reduced response to bradykinin or the Ca2+ ionophore A23187. With multiple cellular passages, used to mimick aging, the basal production of cyclic GMP remained stable during the first passage, to decrease moderately after one month (4th passage). By contrast, the response to bradykinin was reduced as of the second passage, to remain stable thereafter. In cultured aortic endothelial cells, an increase in cellular density was accompanied by a reduced number of active eNOS-site, as well as a reduction of NO production in the response to both bradykinin and A23187. These results suggest that both the increased cellular density and cell senescence explain the endothelial dysfunction during regeneration. They permit a better understanding of the changes in vascular reactivity in the course of endothelial regeneration, and of its pathological consequences.