Rat IGF-I mRNAs contain two different 5'-UTR sequences as a result of alternate splicing of leader exons. Using a combination of solution hybridization/RNase protection and primer extension assays, we have mapped the transcriptional start sites in these leader exons. There appear to be three putative transcription start sites in exon 1 spread over a 140-bp region, the most upstream of which defines a 381 bp-long exon 1. There appear to be three distinct start sites in exon 2, the most upstream of which defines a greater than 770 bp-long exon 2. The two downstream start sites in exon 1 together account for approximately 70% of IGF-I gene expression in adult rat liver. Essentially all of the remaining IGF-I gene expression comes from the second start site in exon 2. Rat IGF-I gene transcription may therefore be regulated by two distinct promoter regions, a disperse promoter for exon 1, with several transcription initiation sites, and a more typical promoter region for exon 2, which controls transcription initiation from a discrete region.