Wound healing involves matrix deposition, angiogenesis, and new tissue growth. Cellular activity during healing is related to tissue oxygen levels. Since wound healing requires oxygen, the purpose of this study was to investigate the effects of hyperbaric oxygen (HBO) on cells involved in wound healing. Cultured endothelial cells and fibroblasts were exposed to HBO. The effect of varied partial pressure, oxygen saturation, and duration and frequency of exposure to HBO on cell proliferation was determined by H-3-labeled thymidine incorporation, HBO causes an increase in the partial pressure of oxygen in the medium of cultured cells, leading to increased endothelial cell and fibroblast proliferation. Increased endothelial cell proliferation occurred after 15 min of HBO. Fibroblasts required 120 min of HBO to produce a response. A second exposure to HBO on the same day produced no additional increase in cell proliferation. A 120-min HBO exposure stimulated fibroblast proliferation for 72 h after the exposure. An increase in pressure from 2.4 to 4.0 atmospheres absolute did not enhance the proliferative response. These studies begin to elucidate the effects of HBO on cells involved in wound healing and establish a scientific foundation upon which to develop more efficacious and cost-effective HBO therapeutic protocols.