Activation of endothelial nitric oxide synthase (eNOS) and subsequent nitric oxide production (NO) are events that mediate the effect of important angiogenic, vasopermeability, and vasorelaxation factors, including vascular endothelial growth factor (VEGF), bradykinin (BK), and acetylcholine (ACh). The N-terminal 16-kDa fragment of prolactin (16K-PRL) acts on endothelial cells to inhibit angiogenesis both in vivo and in vitro. Here, we show that 16K-PRL inhibits VEGF-induced eNOS activation in endothelial cells. Inhibition of eNOS activation may mediate the antiangiogenic properties of 16K-PRL, because the NO donor (Z)-1-[2-(2-aminoethyl)- N-(2-ammonio-ethyl)amino]diazen-1-ium-1,2-diolate (DETANONOate) prevented 16K-PRL from blocking the VEGF-induced proliferation of endothelial cells. In addition, 16K-PRL inhibited eNOS activation by BK and blocked the BK-evoked transient increase in intracellular Ca(2+) in endothelial cells. This finding suggests that 16K-PRL interferes with the mobilization of intracellular Ca(2+), thereby inhibiting the Ca(2+)-dependent activation of eNOS. Blockage of eNOS activation can lead to inhibition of vasodilation. Consistently, 16K-PRL inhibited BK-induced relaxation of coronary vessels in isolated perfused guinea pig hearts. Moreover, 16K-PRL inhibited eNOS activation induced by ACh, and this action resulted in the inhibition of both ACh-evoked relaxation of coronary vessels in isolated perfused rat hearts and ACh-induced, endothelium-dependent relaxation of rat aortic segments. In conclusion, 16K-PRL can block the Ca(2+)-mediated activation of eNOS by three different vasoactive substances, and this action results in the inhibition of both angiogenesis and vasorelaxation.