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The aim of this study was to determine whether different signal transduction mechanisms underlie the Ca2+sensitizing effects of guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and receptor agonists on β‐escin‐skinned smooth muscle of rabbit mesenteric artery.In the homogenate of the β‐escin‐skinned arterial strip, C3 exoenzyme of Clostridium botulinumcatalyzed the [32P]‐ADP‐ribosylation of only one protein that had the same molecular mass as the protein detected in Western blots with anti‐rhop21 antibody. Pretreatment of preparations with C3 resulted in great inhibition of GTPγS‐induced Ca2+sensitization, although the effect of GTPγS at higher concentrations (30 μM) was not completely blocked by this treatment. In contrast, the enhancement by phenylephrine and histamine, in the presence of guanosine 5′‐triphosphate, of the Ca2+‐induced contraction was not affected by C3 pretreatment.The protein kinase C (PKC) inhibitors calphostin C and staurosporine completely eliminated the enhancement by phorbol ester 12,13‐dibutyrate of the Ca2+‐induced contraction. However, these PKC inhibitors had no effect on GTPγS‐ and receptor agonist‐induced Ca2+sensitization.The tyrosine kinase inhibitors genistein and tyrphostin 25 caused an irreversible and complete block of the enhancement by GTPγS of the Ca2+‐induced contraction without affecting this Ca2+contraction. The inactive genistein analogue daidzein did not modify the effect of GTPγS. The Ca2+sensitizing effects of phenylephrine and histamine were also blocked by these tyrosine kinase inhibitors.These results suggest that rhop21 predominantly mediates GTPγS‐induced Ca2+sensitization of β‐escin‐skinned smooth muscle of rabbit mesenteric artery, while the Ca2+sensitizing actions of heterotrimeric G protein‐coupled receptor agonists do not involve this small G protein. However, it seems that tyrosine phosphorylation, but not PKC activation, plays an important role in both of the rhop21 protein‐ and heterotrimeric G protein‐mediated Ca2+sensitization mechanisms.