Upregulation of insulin-like growth factor (IGF)-II expression has been reported for a variety of childhood and adulthood tumors. We determined IGF-II gene promoter usage in human cancerous and benign tissues by semiquantitative RT;PCR using P1-P4-specific primers. Although the human IGF-II gene structure is commonly thought to consist of nine exons and four promoters, we detected substantial utilization of a previously reported exon 4b, which is down-stream of exon 4. Thus, exon 4b was intensively studied using 4b-specific primers, IGF-II gene promoter usage is highly variable in malignant and benign breast, prostate, and bladder tissues. While a majority of samples utilized P2-P4 promoters in a variety of combinations, when quantitated, P3 and P4 promoters were much more active than P2 promoter. This study not only demonstrated that IGF-II gene promoter usage is highly variable in malignant and benign tissues, but suggested that alternatively spliced exon 4b should be recognized as a 10th exon.