Insulin-like growth factor I (IGF-I) promoter activity was characterized in C6, GH3, OVCAR-3, and Chinese hamster ovary (CHO) cells. Maximal exon 1 promoter activity was present in the region extending from -133 to +362 (where +1 is the first transcription start site). Promoter activity was higher in the +75/+362 fragment, which contains exon 1 transcription start sites 3 and 4, than in the -133/+74 fragment, which contains exon 1 transcription start sites 1 and 2. Promoter activity was also observed in constructs containing sequences from -133 to +192, which includes start sites 1, 2, and 3. Inclusion of sequences upstream of -133 inhibited exon 1 proximal promoter activity in a cell type-specific manner. Exon 2 promoter activity was observed in all cell lines with a construct containing 73 bp of 5'-flanking sequence and 44 bp of exon 2. Exon 2 promoter activity was abolished when only 36 bp of 5'-flanking sequence and 44 bp of exon 2 were present, suggesting that an essential minimal promoter element(s) is contained within the -73 to -36 region. A putative CACCC box was observed within this region at -53. Upstream sequence regulated exon 2 promoter activity in a cell type-specific manner. Electrophoretic mobility shift assays revealed a single specifically bound band when the +75/+362 fragment of the exon 1 promoter was used with nuclear extracts from C6 and GH3 cells. Multiple specifically bound bands with slower mobility were observed when the -236/+44 exon 2 promoter fragment was incubated with C6, GH3, CHO, and OVCAR-3 cell nuclear extracts. The exon 1 and exon 2 promoter regions were able to inhibit each other's binding in electrophoretic mobility shift assay using GH3 cell and OVCAR-3 cell nuclear extracts, respectively. Oligonucleotides containing consensus activating protein-1 (AP-1) and AP-3 sequences inhibited exon 1 promoter binding by GH3 cell nuclear extracts. AP-2 and AP-3 sites inhibited exon 2 promoter binding. Our data suggest that the sequence surrounding and including start site 3 in exon 1 functions as a minimal independent promoter. The minimal exon 2 promoter is contained within the 73 bp upstream and 44 bp downstream of the transcription start site cluster. These minimal promoters contain similar and distinct elements that are important for basal transcription. Upstream sequences may contain cell type-specific silencer elements.