Insulin-like growth factor-II (IGF-II) mRNA exists as multiple transcript size classes, such as 6.0, 5.3, 4.9, 3.2, and 2.2 kb mRNAs in various human tissues. Three different promoters, 2 different polyadenylation sites, and alternative splicing are involved in producing these multiple transcripts. Initiation of transcription at the 3 different promoters results in multiple mRNAs which contain identical coding regions but different 5'-untranslated regions (5'-UTRs). The first promoter is thought to direct expression of 5.3 kb mRNA in adult human liver. The second promoter region directs expression of 6.0, 3.2, and 2.2 kb mRNAs in human fetal tissues and several adult nonliver tissues. The third promoter specifies transcription of a 4.9 kb mRNA in various tissues. We isolated and sequenced a cDNA clone (pIGF-II-1-70) from a human placental cDNA library, which contains the IGF-II coding region and the 5'-UTR associated with the third promoter. By using a 5'-UTR-specific probe from the clone, we found that this third 5'-UTR is contained in the IGF-II mRNA of 2.2 kb and is absent in the 3.2 kb IGF-II mRNA. We also found an 0.9 kb transcript expressed in placenta, which hybridized strongly to the third 5'-UTR specific probe but not to IGF-II coding region probes. This finding might indicate the existence of an mRNA encoding an IGF-II-associated peptide.