Testosterone stimulates the expression of IGF-I 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-I mRNA levels in HepG2 and LNCaP cells and stimulated the activity of reporter genes controlled by 1.6 kb of the upstream promoter of the human IGF-I 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 androgen receptor (AR)-binding sites from the Pem1 gene. Reporter genes incorporating these sequences were strongly stimulated by androgens. Each of the androgen-responsive elements (AREs) 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 glucocorticoid receptor. Collectively, these findings indicate the presence of two AREs within the IGF-I upstream promoter that act in cis to activate IGF-I expression. These AREs seem likely to contribute to the up-regulation of the IGF-I gene in prostate tissues, HepG2 cells, and potentially other tissues.