Testosterone stimulates the expression of insulin-like growth factor-1 (IGF-1) in cells and tissues that include prostate, muscle and muscle satellite cells, and the uterus. Here, the molecular mechanisms of this effect of testosterone were explored. Testosterone increased IGF-1 mRNA levels in HepG2 and LNCaP cells and stimulated the activity of reporter genes controlled by 1.6 kb of the upstream promotor of the human IGF-1 gene. An androgen-responsive region that was located between -1320 and -1420 bases upstream of the first codon was identified by truncation studies. The androgen-responsive region was found to contain two sequences resembling known AR binding sites from the Pem1 gene. Reporter genes incorporating these sequences were strongly stimulated by androgens. Each of the androgen-responsive elements bound recombinant AR-DNA binding domain in gel-shift experiments; binding was greatly enhanced by sequences flanking the apparent AR-binding half-sites. Testosterone induced recruitment of AR to sequences of genomic DNA containing these AREs. The two AREs were activated 5-fold more by AR than GR. Collectively, these findings indicate the presence of two AREs within the IGF-1 upstream promotor that act in cis to activate IGF-1 expression. These AREs seem likely to contribute to the upregulation of the IGF-1 gene in prostate tissues, HepG2 cells, and potentially other tissues.