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 somatomedin 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 serum-free 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 immuno-reactive somatomedin eluted from Sephacryl S-200 in two major peaks (150,000 and 8,000 mol wt). The higher molecular weight peak is similar to the one observed when whole serum was used. These studies provide a model system for studying the humoral and nonhumoral factors that control the biosynthesis of somatomedin by human tissues. Since immunoreactive somatomedin production may be a rate-limiting factor for fibroblast growth, the delineation of the hormonal control of somatomedin production should lead to a better understanding of the mechanisms controlling human fibroblast growth.