The mechanisms by which calcium modulates diastolic depolarization (DD) in sheep cardiac Purkinje fibers were studied in vitro. Increasing [Ca]o from 2.7 mM to 10.8 mM increased both the slope and amplitude of DD, induced oscillatory potentials (V(os)), and prolonged depolarization (V(ex)). The steepening of DD occurred even in the absence of an obvious V(os). The increase in DD amplitude was due both to an increase in the maximum diastolic potential and to a less negative steady-state level. At constant [Ca]o, increasing the driving rate had effects similar to those induced by increasing [Ca]o. The increase in DD slope and amplitude was least at the slowest rates and leveled off at the fastest rates in high [Ca]o. Lowering [Ca]o decreased DD slope and amplitude, but spontaneous activity could be present during interruption of the drive. In slowly driven fibers, increasing [Ca]o to 10.8 mM initially shifted the maximum diastolic potential and steady state DD to more negative values, and subsequently shifted the latter (but not the former) to less negative values. On recovery, a transient depolarization occurred. Quiescent fibers exposed to high [Ca]o also underwent a transient hyperpolarization and a subsequent depolarization, whereas reciprocal effects occurred when [Ca]o was lowered. It is concluded that [Ca]o modulates DD through several different mechanisms and that most (but not all) modifications induced are brought about by changes in [Ca]i.