The effects of 10(-5) M trans-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeacetamidel (U-50488H), a kappa-opioid receptor agonist, on cytosolic Ca2+ concentration ([Ca2+]i) and the [Ca2+]i transient in quiescent and electrically stimulated rat ventricular myocytes, respectively, were determined after the cells had been pretreated with pertussis toxin (PTX) or a tyrosine kinase inhibitor (genistein or tyrphostin). The [Ca2+]i was determined with a spectrofluorometric method, with fura 2 as Ca2+ indicator. U-50488H at 10(-5) M itself induced a [Ca2+]i transient in the quiescent cells but inhibited the [Ca2+]i transient in electrically stimulated cells. The effects of 10(-5) M U-50488H on [Ca2+]i, which were blocked by a selective kappa-opioid receptor antagonist, nor-binaltorphimine (10(-6) M), were abolished after pretreatment with PTX (1 microg/ml) for 24 h, but not with genistein (10(-4) M) or tyrphostin (5 x 10(-5) M) for 30 min. 1-[6-[[(17b)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexy l]-1H-pyrrole-2,5-dione (U-73122), an inhibitor of phospholipase C, at 10(-5) M, but not its inactive structural isomer 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexy l]-2,5-pyrrolidinedione (U-73343), also blocked the Ca2+ responses to U-50488H. The results indicate that activation of phospholipase C on kappa-opioid receptor stimulation is via PTX-sensitive G proteins but does not involve protein tyrosine phosphorylation.