1. Effects of guanosine triphosphate (GTP) and guanosine 5'-o-(3-thio)triphosphate (GTP gamma S) on mechanical properties of skinned smooth muscle tissues of the rabbit mesenteric artery were investigated. 2. In skinned muscle tissues prepared by saponin, GTP (above 100 microM) and GTP gamma S (above 1 microM) enhanced the Ca2+-induced contraction (0.3 microM-Ca2+ buffered with 2 mM-EGTA) in the presence of 1 microM-ionomycin, a depletor of stored Ca2+. The concentration-response (pCa-tension) relationship observed in the presence of 10 microM-GTP gamma S shifted to the left with no change in the maximum response evoked by 10 microM-Ca2+. The action of GTP was reversible but that of GTP gamma S was not. 3. The enhancement of the Ca2+-induced contraction by GTP gamma S occurred with increases in the phosphorylation of myosin light chain and in the shortening velocity as measured with the slack test. 4. GTP gamma S had no effect on the Ca2+-independent contraction of skinned muscle tissues evoked by MgATP in Ca2+-free solution (4 mM-EGTA), following treatment with rigor solution containing adenosine 5'-o-(3-thio)triphosphate (ATP gamma S). 5. The present results indicate that GTP and GTP gamma S enhance the Ca2+-induced contraction in skinned muscle tissues due to increase in the Ca2+ sensitivity of contractile proteins. These enhancing actions of guanosine nucleotides on contractile proteins are discussed in comparison to those of protein kinase C.