We hypothesized that the Src family tyrosine kinases (STKs) are involved in the up-regulation of arginase and inducible nitric oxide synthase (iNOS) expression in response to inflammatory stimuli in pulmonary endothelial cells. Treatment of bovine pulmonary arterial endothelial cells (bPAEC) with lipopolysaccharide and tumor necrosis factor- (L/T) resulted in increased urea and NO production and this increase in urea and NO production was inhibited by the STK inhibitor PP1 (10 µM). The STK inhibitors, PP2 (10 µM) and herbimycin A (10 µM), also prevented the L/T-induced expression of both arginase II and iNOS mRNA in bPAEC. Taken together the data demonstrate a central role of the STK in the up-regulation of both arginase II and iNOS in bPAEC in response to L/T treatment. To identify the specific kinase(s) required for the induction of urea and NO production, we studied human pulmonary microvascular endothelial cells (hPMVEC) so that siRNA techniques could be employed. We found that hPMVEC express fyn, yes, c-src, lyn, and blk, and that the protein expression of fyn, yes, c-src, and lyn could be inhibited using specific siRNA. The siRNA targeting fyn prevented the cytokine-induced increase in urea and NO production, while siRNAs specifically targeting yes, c-src or lyn had no appreciable effect on cytokine-induced urea and NO production. These findings support our hypothesis that inflammatory stimuli lead to increased urea and NO production through a STK-mediated pathway. Furthermore, these results indicate that the STK fyn plays a critical role in this process.