We examined the hypothesis that vacuolation is a normal feature of interstitial cells of Cajal. With the zinc iodide-osmic acid stain, and using light microscopy, we examined the interstitial cells in Stach's plexus on the submucosal surface of the circular muscle layer of the colon in the rat. We subjected tissues to seven treatments designed to produce different degrees of damage from hypoxia. In the least-damaged colons (fixed immediately after dissection) about 20% of interstitial cells showed vacuoles. In the most damaged colons (immersed for 30 min in Krebs' solution equilibrated with 95% N2/5% CO2 at 37 degrees C) about 96% of cells showed vacuoles. Less drastic treatments produced fewer vacuoles, but even colons immersed for 30 min in Krebs' solution equilibrated with 95% O2/5% CO2 at 37 degrees C showed vacuolation in about 28% of cells. The light microscopic impression was confirmed by electronmicroscopy, but cells subjected to even the most severe treatment retained a recognizable ultrastructure. Vacuolation in interstitial cells is probably artifactual, a consequence of hypoxia.