Cyclical periods of depolarization (slow waves) underlie peristaltic contractions involved in mixing and emptying of contents in the gastric antrum. Slow waves originate from a myenteric network of interstitial cells of Cajal (ICC-MY). In this study we have visualized the sequence and propagation of Ca2+ transients associated with pacemaker potentials in the ICC network and longitudinal (LM) and circular muscle (CM) layers of the isolated guinea-pig gastric antrum. Gastric antrum was dissected to reveal the ICC-MY network, loaded with Fluo-4 AM and activity was monitored at 37°C. Ca2+ waves propagated throughout the ICC-MY network at an average velocity of 3.24 ± 0.12 mm s−1 at a frequency of 4.87 ± 0.16 cycles min−1 (n = 4). The propagation of the Ca2+ wave often appeared ‘step-like’, with separate regions of the network being activated after variable delays. The direction of propagation was highly variable (Δ angle of propagation 44.3 ± 10.9 deg per cycle) and was not confined to the axes of the longitudinal or circular muscle. Ca2+ waves appeared to spread out radially from the site of initiation. The initiating Ca2+ wave in ICC-MY was correlated to secondary Ca2+ waves in intramuscular interstial cells of Cajal, ICC-IM, and smooth muscle cells, and the local distortion (contraction) in a field of view. TTX (1 μm) had little effect on slow wave or pacemaker potential activity, but 2-APB (50 μm) blocked all Ca2+ waves, indicating a pivotal role for intracellular Ca2+ stores. Nicardipine (2 μm) eliminated the Ca2+ transient generated by smooth muscle, but did not affect the fast upstroke associated with ICC-MY. These results indicate that slow waves follow a sequence of activation, beginning with the ICC-MY and ICC-IM network, followed later by a sustained Ca2+ transient in the muscle layers that is responsible for contraction.