1. The effects of the serine/threonine protein phosphatases (PP) type 1 and 2A on L-type Ca2+ current (ICa) and the intracellular [Ca2+]i transient were examined in rat ventricular myocytes. ICa was measured under voltage clamp using patch-type microelectrodes in the whole-cell mode with the cells in a steady state of sarcoplasmic reticulum (SR) Ca2+ loading. [Ca2+]i transients were measured simultaneously using the fluorescent Ca2+ indicator indo-1 (50 microM) which was added to the pipette filling solution along with PP-1 or PP-2A (4 units ml-1). 2. PP-1 had no effect on the ICa-V relationship but decreased the [Ca2+]i-voltage relationship (by 43% at 0 mV). PP-2A decreased both ICa-V (by 26% at 0 mV) and the [Ca2+]i transient-voltage (by 65% at 0 mV). Excitation-contraction coupling gain, defined as (delta [Ca2+]i/ICa), was decreased to 43% of control by PP-1 and to 29% of control by PP-2A at-28 mV. 3. Diastolic [Ca2+]i (i.e.[Ca2+]i measured immediately before each voltage clamp pulse) was not altered by PP-1 or PP-2A and neither phosphatase changed steady-state SR Ca2+ content, as measured with caffeine. 4. We conclude that the reduced [Ca2+]i transient following the application of PP-1 was due to reduced SR Ca2+ release channel activity. The effects of PP-2A, while more broadly based, were still consistent with a decrease in SR Ca2+ release channel activity. 5. Our experiments, combined with recent experiments by others, suggest that the basal state of contractility in heart is dynamically regulated by dephosphorylation and phosphorylation of the SR Ca2+ release channel.