1. We used confocal Ca2+ imaging and fluo-3 to investigate the transition of localized Ca2+ releases induced by focal caffeine stimulation into propagating Ca2+ waves in isolated rat ventricular myocytes. 2. Self-sustaining Ca2+ waves could be initiated when the cellular Ca2+ load was increased by elevating the extracellular [Ca2+] ([Ca2+]0) and they could also be initiated at normal Ca2+ loads when the sensitivity of the release sites to cytosolic Ca2+ was enhanced by low doses of caffeine. When we prevented the accumulation of extra Ca2+ in the luminal compartment of the sarcoplasmic reticulum (SR) with thapsigargin, focal caffeine pulses failed to trigger self-sustaining Ca2+ waves on elevation of [Ca2+]0. Inhibition of SR Ca2+ uptake by thapsigargin in cells already preloaded with Ca2+ above normal levels did not prevent local Ca2+ elevations from triggering propagating waves. Moreover, wave velocity increased by 20%. Tetracaine (0.75 nM) caused transient complete inhibition of both local and propagating Ca2+ signals, followed by full recovery of the responses due to increased SR Ca2+ accumulation. 3. Computer simulations using a numerical model with spatially distinct Ca2+ release sites suggested that increased amount of releasable Ca2+ might not be sufficient to generate self-sustaining Ca2+ waves under conditions of Ca2+ overload unless the threshold of release site Ca2+ activation was set at relatively low levels (< 1.5 μM). 4. We conclude that the potentiation of SR Ca2+ release channels by luminal Ca2+ is an important factor in Ca2+ wave generation. Wave propagation does not require the translocation of Ca2+ from the spreading wave front into the SR. Instead, it relies on luminal Ca2+ sensitizing Ca2+ release channels to cytosolic Ca2+.