The hypothesis was investigated that NG-hydroxy-L-arginine (L-HOArg) is an intermediate in the biosynthesis of nitric oxide (.NO) from L-arginine (L-Arg) by the constitutive .NO synthase (NOS) present in endothelial cells (ECs). When infused through a column of bovine aortic ECs on beads, either L-HOArg or D-HOArg (1-10 microM) substantially potentiated relaxations of the bioassay tissues to .NO released from the cells by ADP or bradykinin, and this effect was abolished by coinfusions of NG-nitro-L-arginine (L-NO2Arg) methyl ester (10 microM) or NG-monomethyl-L-arginine (L-MeArg; 30 microM). Both L-HOArg and D-HOArg, irrespective of the presence of ECs, also potentiated relaxations induced by authentic .NO, but not glyceryl trinitrate. This was due to a rapid chemical reaction of either isomer with .NO, resulting in the formation of a potent and more stable vasodilator. When infusions of L-HOArg (3 microM) were consequently made in the presence of D-HOArg (10 microM), the L-isomer no longer had any effect on relaxations induced by authentic .NO, but significantly increased the stimulated release of .NO from the column of ECs. The conclusion that L-HOArg is a substrate for the constitutive NOS in cultured ECs was strongly supported by the L-NO2Arg-sensitive conversion of L-HOArg, but not D-HOArg, to .NO by NOS preparations from these cells. Interestingly, cultured ECs produced from L-HOArg (greater than or equal to 3 microM), but not D-HOArg, a stable vasodilator, the effects of which were inhibited by oxyhemoglobin (0.3-3 microM). However, the formation of this substance was not prevented by L-NO2Arg methyl ester (10 microM) or L-MeArg (10-100 microM), suggesting an enzymatic pathway different from NOS.