The proliferative effects of insulin on infrapopliteal vascular smooth muscle cells (VSMCs) have been established. We examined the effect of hypoxia in the presence and absence of insulin on the proliferation and migration of human diabetic infrapopliteal VSMCs in vitro. VSMCs isolated from the infrapopliteal arteries of male diabetic patients of identical disease and clinical patterns undergoing below-knee amputation were harvested and grown to subconfluence. Cells were then exposed to control medium (M199/1% fetal bovine serum/2% antibiotic-antimycotic) or control medium with 100 ng/mL insulin in oxygen concentrations of 17% (normoxia), 5%, and 1%. Cellular proliferation was assayed using [methyl-3H]-thymidine incorporation. Migration assays were performed using the Corning Costar Transwell system. Lactate dehydrogenase was assayed and compared among groups as a marker for cytotoxicity. VSMCs in normoxic conditions (17%) had a significant increase in both proliferation (100 +/- 6.5% vs. 124 +/- 4.7%, p = 0.007) and migration [73.2 +/- 9.3 vs. 118.1 +/- 14.9 cells/4 high-power fields (HPF), p = 0.03] when exposed to insulin. Of cells exposed to insulin, those at both 5% (75.9 +/- 7.9%, p = 0.0001) and 1% (73.6 +/- 4%, p < 0.0001) hypoxia proliferated at a significantly decreased rate compared with cells at normoxia (124 +/- 4.7%). Migration of these insulin-exposed cells was significantly decreased at 1% hypoxia (63.1 +/- 9.0 cells/4HPF) compared to those at normoxia (118.1 +/- 14.9 cells/4HPF, p = 0.006) and 5% hypoxia (101.2 +/- 10.0 cells/4HPF, p = 0.01). There were no significant differences in migration between cells at normoxia and 5% hypoxia. Finally, hypoxia and insulin exerted no significant effect on cytotoxicity. The proliferative and promigratory effects of insulin on diabetic VSMCs are attenuated in hypoxic conditions in a manner unrelated to cytotoxicity.