Most insulin-like growth factor-I (IGF-I) transcripts are initiated in exon 1, but mechanisms of regulation are not well understood. Since potential Sp1 sites are found in footprinted regions within ∼360 bp upstream and downstream from the major initiation sites in exon 1, we explored the involvement of Sp1 and Sp3 in regulation of IGF-I expression. Gel shift assays showed strong Sp1 binding to the downstream site, but binding to the upstream site was weak; Sp1 bound to a CCTGCCCA sequence in downstream footprint region V, and Sp3 binding was centered on the same sequence. IGF-I basal promoter constructs containing a mutation in the downstream Sp1 site exhibited a 32% decrease in expression in CHO cells and a 75% decrease in HepG2 cells, indicating the importance of Sp1 for expression in vivo. Sp1 and Sp3 expression vectors provided three- to five-fold stimulation of wild-type IGF-I constructs, but had little effect on a construct containing a mutation in the downstream Sp1 site, and Sp1 had comparable effects in Drosophila SL2 cells. IGF-I heterologous promoter constructs exhibited similar responses: in both SL2 cells and CHO cells, stimulation by Sp1 was enhanced with constructs containing downstream region V. Since Sp1 also stimulated expression of concatamers of putative cis-acting sites fused to the SV40 promoter–enhancer in pGL3, the results in combination indicate that the presence of IGF-I region V is sufficient to permit stimulation by Sp1. Conclusion: Sp1 and related factors may play an important role in the regulation of IGF-I gene transcription, through interactions with region V downstream from the major initiation sites in exon 1.