The present experiments were designed to investigate the mechanisms involved in the contractile responses evoked by KCl, added either isoosmotically or hyperosmotically, in the rat uterus. Exposure of uterine strips to a Ca(2+)-free, 3 mM EGTA-containing solution abolished the responses induced by isoosmotic KCl solutions. Conversely, addition of hyperosmolar KCl induced concentration-dependent tonic responses in a Ca(2+)-free, 3 mM EGTA-containing solution. The maximum increase in tension was reached with 210 mM K+. The response to hyperosmotic K+ was unaffected by previous depletion of intracellular Ca2+ stores with oxytocin (1 microM), by inhibition of refilling of the intracellular Ca2+ stores using cyclopiazonic acid (10 microM) or by increasing the concentration of EGTA in the medium to 10 mM. Sucrose and mannitol (60-420 mM) induced concentration-dependent sustained contractions which were not reproducible and were significantly smaller in size than those evoked by the maximally effective concentration of hyperosmotic K+ (210 mM). The contraction induced by hyperosmotic K+ in Ca(2+)-free solution was not altered by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, 100 microM), the Ca2+/calmodulin protein kinase II inhibitor 1-[N,O-bis(1,5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenyl piperazine (KN-62, 10 microM) or the tyrosine kinase inhibitor genistein (10 microM). The protein kinase C inhibitor calphostin C (1-3 microM) failed to modify the K(+)-effect curve, which was however partially inhibited in the presence of the non-selective protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2 methylpiperazine dihydrochloride (H-7, 3-100 microM). The protein kinase inhibitor staurosporine (30-300 nM) depressed the contraction induced by hyperosmolar K+ in a concentration-dependent manner. The contraction induced by sucrose in Ca(2+)-free solution was unaffected by W-7 (100 microM) and KN-62 (10 microM) but was partially reduced by calphostin C (1 microM), H-7 (30 microM), staurosporine (100 nM) and genistein (10 microM). These results suggest that different mechanisms are involved in the responses evoked by isoosmotic and hyperosmotic KCl in the rat uterus. A component of the contraction induced by hypertonic KCl seems mainly independent of both external and internal Ca2+ and of hyperosmolar stress. This contraction is not mediated by protein kinase C, Ca2+/calmodulin-dependent kinases or protein tyrosine kinases but involves activation of other, at the present unknown, staurosporine-sensitive protein kinase(s).