This study investigated the interaction between L-type Ca2+ current (ICaL) and Ca2+ release from the sarcoplasmic reticulum (SRCR) in whole cell voltage-clamped guinea pig ventricular myocytes. Quasiphysiological cation solutions (Nao+:KI+) were used for most experiments. In control conditions, there was no obvious interaction between ICaL and SRCR. In isoproterenol, activation of ICaL from voltages between -70 and -50 mV reduced the amplitude and accelerated the decay of the current. Short (50 ms), small-amplitude voltage steps applied 60 or 510 ms before stimulating ICaL inhibited and facilitated the current, respectively. These changes were blocked by ryanodine. Low-voltage activated currents such as T-type Ca2+ current, TTX-sensitive ICa (ICaTTX), or "slip mode" Ca2+ conductance via INa+ were not responsible for low-voltage SRCR. However, L-type Ca2+ currents could be distinguished at voltages as negative as -45 mV. It is concluded that in the presence of isoproterenol, Ca2+ release from the SR at negative potentials is due to activation of L-type Ca2+ channels.