There is evidence that anabolic steroids, which are derived from testosterone and have markedly less androgenic activity, promote tissue growth and enhance tissue repair; however, the mechanisms involved in their anabolic activities remain unclear. In this report, we measured the effect of the anabolic steroid stanozolol on cell replication and collagen synthesis in cultures of adult human dermal fibroblasts. Stanozolol (0.625–5 μg per ml) had no effect on fibroblast replication and cell viability (p = 0.764) but enhanced collagen synthesis (p < 0.01) in a dose-dependent manner (r = 0.907). Stanozolol also increased (by 2-fold) the mRNA levels of α1(I) and α1(III) procollagen and, to a similar extent, upregulated transforming growth factor-β1 (TGF-β1) mRNA and peptide levels (p < 0.001). There was no stimulation of collagen synthesis by testosterone. The stimulatory effects of stanozolol on collagen synthesis were blocked by a TGF-β1 anti-sense oligonucleotide, by antibodies to TGF-β, and in dermal fibroblast cultures derived from TGF-β 1 knockout mice. We conclude that collagen synthesis is increased by the anabolic steroid stanozolol and that, for the most part, this effect is due to TGF-β1. These findings point to a novel mechanism of action of anabolic steroids.