In beta-escin-permeabilized cultured pig aortic smooth muscle cells GTP gamma S dose-dependently enhances Ca(2+)-induced, wortmannin-sensitive phosphorylation of 20 kDa myosin light chain (MLC20). GTP gamma S does not potentiate thiophosphorylation of MLC20, but does inhibit its dephosphorylation. Pretreatment with C. botulinum exotoxin C3, which specifically ADP-ribosylates and inactivates the rho family of the small molecular weight G proteins, completely abolishes the effects of GTP gamma S. These results indicate that rho is involved in the GTP gamma S-induced enhancement of Ca(2+)-dependent MLC20 phosphorylation in aortic smooth muscle cells, and strongly suggest that this effect of rho is due to inhibition of protein phosphatase activity toward MLC20.