The effects of ascorbic acid and hydrocortisone on activity of prolyl hydroxylase in fibroblasts from keloid and normal human dermis were investigated and compared to the effects of these agents on collagen synthesis. Prolyl hydroxylase activity in normal fibroblasts grown to confluency in 1.5 microM hydrocortisone was approximately half that of cells grown without the steroid. The concentration of hydrocortisone effective in reducing enzyme activity was the same as that for reducing the rate of collagen synthesis; a half-maximal effect on both parameters was achieved at 10(-7) M. Hydrocortisone lowered enzyme activity through most of the culture cycle. Fibroblasts derived from keloids were significantly less subject to hydrocortisone-mediated reduction of prolyl hydroxylase activity and rate of collagen synthesis. This difference between keloid and normal cells was dependent on the simultaneous presence of ascorbic acid and hydrocortisone. These data suggest that the defect in wound healing that results in keloid formation is associated with a change in a regulatory mechanism that controls the rate of collagen synthesis and is sensitive to physiological levels of hydrocortisone. Continuous culture of fibroblasts in medium supplemented with ascorbic acid also lowered prolyl hydroxylase activity. Unlike the effect of hydrocortisone, growth in ascorbate increased the rate of collagen synthesis and affected keloid and normal strains equally.