The availability of intracellular reduced thiols, such as L-cysteine or glutathione (GSH), may be critically important for the biosynthesis of endothelium-derived relaxing factor (EDRF). We have, therefore, investigated the effects of various sulfhydryl (SH) reagents, such as 1-chloro-2,4-dinitrobenzene (CDNB), diamide, 2,2'-dithiodipyridine (DTDP), or N-ethyl-maleimide (NEM), on the release of EDRF from cultured endothelial cells. None of the SH reagents tested affected the flow-induced EDRF release, but DTDP and NEM inhibited the release of EDRF stimulated by ADP, ionomycin, or poly-L-lysine. In contrast, NG-nitro-L-arginine methyl ester, an inhibitor of EDRF biosynthesis, blocked both the flow- and agonist-induced release of EDRF. Although NEM substantially potentiated the flow-induced release of prostacyclin (PGI2), probably due to a blockade of the reacylation of arachidonic acid, it inhibited the stimulated release of PGI2, whereas diamide did not significantly affect either release. Like CDNB or diamide, NEM, but not DTDP, caused a significant decrease in endothelial GSH. In contrast, both NEM and DTDP, but not CDNB or diamide, inhibited the ADP-induced mobilization of intracellular calcium, suggesting that they act on specific target proteins involved in endothelial cell calcium homeostasis rather than intracellular free SH groups. Moreover, the selective inhibition by these two SH reagents of the stimulated release of EDRF implies that a fundamental regulatory difference exists between agonist- and flow-induced EDRF biosynthesis.