The effects of 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), a myosin light chain kinase inhibitor, and (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, on thromboxane A2 receptor-mediated signal transduction were examined in rabbit washed platelets. ML-7 and W-7 at 10-30 microM slightly potentiated the aggregation induced by a thromboxane A2 receptor agonist, 9,11-dideoxy-9 alpha,11 alpha- epoxymethanoprostaglandin F2 alpha (U46619), in spite of their known inhibitory actions. ML-7 and W-7 concentration-dependently enhanced U46619-induced phosphoinositide hydrolysis and the increase in internal free Ca2+ concentration in the presence or absence of external Ca2+. While ML-7 and W-7 inhibited basal GTPase activity, they augmented U46619-induced activation of GTPase in a concentration-dependent manner. The present results suggest that ML-7 and W-7 enhance thromboxane A2 receptor-mediated signal transduction at the receptor/G protein coupling, leading to the enhancement of phosphoinositide hydrolysis and Ca2+ mobilization, independently of the inhibition of myosin light chain kinase or calmodulin.