Intracellular calcium [Ca2+]i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca2+]i transient followed by a steady-state increase as the characteristic [Ca2+]i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca2+]i signal in single cells. The distribution of [Ca2+]i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca2+]i present in the subplasmalemmal space and in one cell pole. [Ca2+]i gradients within these regions were not constant but showed temporal changes in the form of [Ca2+]i oscillations and spatial changes in the form of [Ca2+]i waves. [Ca2+]i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca2+] and had a mean frequency of 12.6 +/- 1.1 oscillations per min. The baseline [Ca2+]i was 171 +/- 13 nM and the mean oscillation amplitude was 194 +/- 12 nM. Generation of [Ca2+]i waves was also independent of influx of extracellular Ca2+. [Ca2+]i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +/- 3 microns per sec (n = 6). Increases of [Ca2+]i induced by different agonists were encoded into changes of baseline [Ca2+]i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca2+]i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca2+]i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca2+]i oscillations.