While the functions of several G protein alpha subunits such as alpha(s( and alpha(q) are relatively well understood, the action of others such as alpha13 remain largely undefined. Because of recent interest in regulation of nitric-oxide synthase (NOS) by G protein-coupled signaling systems and findings that receptors for two proinflammatory substances, thrombin and thromboxane couple to alpha13, we studied the effect of alpha13 on NOS activity in a renal epithelial cell line. We found that stable overexpression of alpha13 or its GTPase-deficient mutant, alpha13Q226L, in a continuous renal epithelial cell line (MCT) increased NOS activity. The increased NOS activity was due to increased expression of the macrophage-inducible form of NOS (iNOS). iNOS protein and activity were not increased in similar cells expressing an activated alpha(s) (alpha(s)Q227L) or were minimally increased in cells expressing activated alpha(i1) (alpha-i1Q204L) and alpha(q) (alpha(q)Q209L), members of the three other G protein alpha chain families. Transient co-expression of alpha13 or alpha13Q226L increased the activity of an iNOS promoter-CAT construct demonstrating that alpha13 increases iNOS expression through transcription. Consequently, alpha13 induces iNOS through a novel mechanism that is distinct from that of other G protein alpha chains and that may mediate the actions of G protein-dependent proinflammatory agents.