We examined the effect of a prolyl hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate (E-DHB), on collagen production, proliferation, and spreading by cultured rabbit keratocytes and subconjunctival fibroblasts. Proliferating cells were incubated with or without E-DHB, and were examined by phase-contrast microscopy and proliferation assays. In addition, wounds were made in confluent cultures and were allowed to heal with or without exposure to E-DHB. Confluent cells were also observed by transmission electron microscopy after exposure to E-DHB (0.2-0.4 mM). Finally, confluent cells were incubated in the presence or absence of E-DHB, after which the concentration of soluble collagen in the culture medium was determined. E-DHB (0.2-0.4 mM) inhibited the growth of rabbit keratocytes and subconjunctival fibroblasts and decreased collagen production without inducing cytotoxicity.. Wounds in the control culture were initially healed by individual migrating cells and later by spreading monolayer sheets and proliferating cells, while E-DHB inhibited cell migration into the wound. Electron microscopy revealed large irregular cisternae of endoplasmic reticulum in E-DHB-treated cells, possibly due to protein accumulation. These findings suggest that collagen production may be closely related to various cellular activities.