Activation of the transient inward current (In) by a rise in intracellular calcium concentration ([Ca2+]i) is believed to be responsible for generating triggered cardiac arrhythmias. In this study, the cellular basis of the rise in [Ca2+]ithat activates I-n and aftercontractions in single rat ventricular myocytes was examined. [Ca2+]iwas measured both indirectly by cell contraction and directly with fura-2. Under conditions that caused steady-state [Ca2+]ito increase (i.e., calcium overload) membrane repolarization after a voltage-clamp depolarization resulted in the appearance of In that was similar in many respects to that observed in muiticellular preparations. This In occurred at the same time that [Ca2+]; spontaneously increased and preceded the aftercontraction by 60–90 msec. However, IT, recorded from a single cell was variable in time course and amplitude (unlike that observed in muiticellular preparations). Examination of cell contraction and digital imaging of fura-2 fluorescence showed that In was often associated with propagating regions of increased [Ca2+]i, which arose from discrete sites of origin within the cell. Apparently synchronous aftercontractions could also be associated with multiple propagating waves of [Ca2+]i. The variation in the time course and amplitude of In in single cells appeared to be due to changes in the location and number of sites of origin for the waves of [Ca2+]i. After the first aftercontraction and I-n, desynchronization of the sites of origin of increased [Ca2+]ioccurred, and this resulted in a decrease in the amplitude of In and an increase in its duration. We conclude that the variability seen in single cells arises from changes in the pattern of spontaneous Ca release. Such phenomena will seriously complicate interpretation of muIticellular data, even when [Ca2+], is measured directly.