We describe the two-dimensional imaging of excitation-induced Ca gradients in isolated myocytes under physiological conditions, using a novel method of flash photography of fluo-3 fluorescence. This method is useful for showing the spatial distribution and reproducibility of rapidly triggered Ca release events, and their relationship to underlying structures. In normal rat myocytes, Ca sparks were evident 6ms after stimulation emerging from around t-tubules, as judged by co-localization with di-8-ANEPPS staining. Gaps in the spark pattern coincided with gaps in di-8-ANEPPS staining. Vacuolar fluo-3 uptake, previously identified as lysosomal, was prominent in some of the gaps, suggesting possible areas of t-tubule turnover. In normal dog myocytes, the beat-to-beat variance of Ca sparks was very low, t-tubular voids were small, and Ca gradients resolved rapidly. In myocytes from dogs with failure induced by rapid pacing, a reduced Ca transient was observed associated with increased areas that were void of sparks and t-tubules, and a greater beat-to-beat spark variance. These abnormalities resulted in a non-uniform spatial distribution of sparks, leading to Ca gradients across the cell that persisted for longer times after stimulation. Such Ca gradients could cause heterogeneous contraction and contribute to contractile failure.