The mechanism of enhancement of the L-type Ca current by a P2-purinergic agonist adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was studied by recording single channel activity from cell-attached patches on rat isolated ventricular cells using patch pipettes containing 110 mM Ba2+. The application of ATP gamma S to the patch membrane through the pipette solution did not affect single channel activity. The addition of ATP gamma S to the bath containing a depolarizing solution was ineffective due to the voltage dependence of the purinergic stimulation. Bath application of ATP gamma S (100 microM) to control 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES) solution increased the amplitude of ensemble average currents both by decreasing the probability of a blank sweep occurring and by increasing the number of openings per non-blank sweep. The single channel conductance (17 pS) was not changed by ATP gamma S. Both activation and inactivation curves were shifted towards hyperpolarized potentials by about 10 mV under P2-purinergic stimulation. Since ATP gamma S increased channel activity when applied via the bath, it must be supposed that a diffusible messenger is involved.