Previous work has shown that small depolarizing pulses produce a beat to beat alternation in the amplitude of the systolic Ca2+ transient in ventricular myocytes. The aim of the present work was to investigate the role of changes of SR Ca2+ content and L-type Ca2+ current in this alternans. As the amplitude of the depolarizing pulse was increased from 10 to 30 mV the magnitude of alternans decreased. Confocal linescan studies showed that this was accompanied by an increase in the number of sites from which Ca2+ waves propagated. A sudden decrease in the depolarisation amplitude resulted in three classes of behaviour: (1) a gradual decrease in Ca2+ transient amplitude before alternans developed accompanied by a loss of SR Ca2+, (2) a gradual increase in Ca2+ transient amplitude before alternans accompanied by a gain of SR Ca2+, and (3) immediate development of alternans with no change of SR content. We conclude that alternans develops if the combination of decreased opening of L-type channels and change of SR Ca2+ content results in spatially fragmented release from the SR as long as there is sufficient Ca2+ in the SR to sustain wave propagation. Potentiation of the opening of the ryanodine receptor (RyR) by low concentrations of caffeine (100 μm) abolished alternans for a few pulses but the alternans then redeveloped once SR Ca2+ content fell to the new threshold for wave propagation. Finally we show evidence that inhibiting L-type Ca2+ current with 200 μm Cd2+ produces alternans by means of a similar fragmentation of the Ca2+ release profile and propagation of mini-waves of Ca2+ release.