A B S T R A C T Human growth hormone (hGH) is known to be a potent stimulator of somatomedin secretion in vivo. The induction of somatomedin by growth hormone has been difficult to study in vitro, however, because no organ containing a high concentration of sQmatomedin has been identified. Because fetal mouse explants have been shown to produce somatomedin in vitro, we have undertaken studies to determine whether postnatal human fibroblast monolayers also produce somatomedin, and if so, whether its production is regulated by other hormones. Quiescent human fibroblasts were exposed to serumfree minimum essential medium, and the medium was assayed for somatomedin concentration using a specific radioimmunoassay for somatomedin-C. A progressive rise in immunoreactive somatomedin to 0.08 U/ml per 105 cells per 24 h was observed over 72 h of incubation. This was an underestimation of the actual concentration of immunoreactive somatomedin since the amount measured following acid treatment was at least fourfold higher than in the untreated medium. Growth hormone stimulated immunoreactive somatomedin production in a dose-dependent manner: 5 ng hGH/ml = 0.1 U/ml per 105 cells; 50 ng hGH/ml = 0.25 U/ml per 105 cells. Platelet-derived growth factor and fibroblast growth factor were also stimulatory, but epidermal growth factor, thyroxine, or cortisol had no effect. Media that had been exposed to human fibroblasts stimulated DNA synthesis in BALB/c 3T3 fibroblasts (a cell type that does not produce somatomedin). Medium-derived immunoreactive somatomedin eluted from Sephacryl S-200 in two major peaks (150,000 and 8,000 mol wt).