The objective of this study was to search for polymorphisms and gene regulatory sequences in the 5' flanking region of the sheep insulin-like growth factor I (IGF-I) gene. PCR-SSCP analysis of the 5' flanking region revealed three banding patterns. Family study indicated that these patterns in mixed breed sheep corresponded with three genotypes (with their frequencies in parentheses) AA (0.70), AB (0.25), and BB (0.05), which arose from a one-locus, two allele (A, B) polymorphism. Genotypic frequencies in 22 purebred Polypay sheep were AA (0.77) and AB (0.23). Calculated frequency of the A allele in Polypays was 0.89. No deviation from Hardy-Weinberg equilibrium was detected in this study. Fragments amplified using DNA from homozygous individuals were sequenced and aligned next to each other. A T to C transition and a G to C transversion were found at positions 179 and 181, respectively, of the amplified PCR product, resulting in recognition sites for Bsp143II and HaeI. Analysis of a fragment of 2,162 base pairs upstream of Exon 1, assembled from sheep ESTs and sequence of our amplified PCR products, revealed a promoter sequence approximately 100 bp downstream of the polymorphic sites. The assembled DNA fragment shared 70% sequence homology between sheep and human. These results suggest that sequence of the 5' flanking region of IGF-I gene and location of the IGF-I promoters are similar in human and sheep.