The rat insulin-like growth factor II (rIGF-II) gene, which exists as a single copy in the genome, is expressed as a multitranscript family of mRNA molecules ranging in size from 4.6 to 1 kilobases. Part of this heterogeneity can be ascribed to the presence of two different promoters, each transcribing alternative 5'-noncoding regions which are spliced to common coding exons. In the present study we use a combination of DNA sequence analysis of the gene, mapping of the mRNA molecules by Northern analysis and ribonuclease protection experiments, and DNA sequence analysis of cDNA clones complementary to different regions of the genome to establish the structure of several rIGF-II mRNA species. These results indicate that RNA heterogeneity also arises from the use of different polyadenylation sites. In addition, a variant 2 kilobases RNA was observed that was colinear with the distal 1700 base pairs of the 3147 base pair long exon 3, and may arise by alternative RNA splicing. These posttranscriptional modifications of RNAs arising from the rIGF-II transcription unit may generate molecules with different functional potential.