1. In rabbit papillary muscles, pretreatment with pertussis toxin (PTX) significantly increased the positive inotropic response to isoprenaline and abolished the inhibitory action of carbachol on the isoprenaline response. 2. Phenylephrine in the presence of propranolol produced a positive inotropic effect and prolonged action potential duration through activation of alpha 1-adrenoceptors. Both of the effects of phenylephrine were significantly enhanced by PTX pretreatment. 3. Accumulation of [3H]inositol monophosphate (IP1) in papillary muscles prelabeled with myo-[3H]inositol was increased by phenylephrine in a concentration-dependent manner, which was antagonized by prazosin. Although PTX pretreatment significantly elevated the basal level of [3H]IP1 formation, the phenylephrine-induced increase in [3H]IP1 formation was unaffected. 4. It is concluded that the cardiac responses to alpha 1-adrenoceptor stimulation studied in these experiments are not transduced by a PTX sensitive G protein (Gi). However, the positive inotropic effect and prolongation of action potential duration mediated by alpha 1-adrenoceptor may be negatively regulated by Gi.