We found low T3 concentrations in rat brown adipocytes differentiated in vitro. This might be due to the high metabolic rate of T3, possibly caused by elevated type III iodothyronine 5-deiodinase activity (5DIII), induced by serum growth factors. We tested the ability of several growth factors to induce 5DIII activity. Epidermal growth factor and basic and acidic fibroblast growth factors produced a strong induction of 5DIII activity (25, 45-, and 50-fold, respectively). This process required gene transcription and de novo protein synthesis. The half-life of 5DIII was approximately 3 h. Heparin was required for full acidic fibroblast growth factor activity. Platelet-derived growth factor, vasopressin, and insulin-like growth factor-I induced lower 5DIII activities (3- to 6-fold). Vasopressin amplified basic fibroblast growth factor and epidermal growth factor inductions when used at submaximal doses. We found a Km of 22.5 nM using T3 as substrate. Although brown adipose tissue has undetectable 5DIII activities in vivo, the present data explain the low T3 concentrations found in cultured rat brown adipocytes and suggest that growth factors, by stimulating 5DIII, may lead to low T3 concentrations and indirectly inhibit the expression of some genes regulated by T3, e.g. the rat uncoupling protein.