Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide that is thought to play a role in fetal growth and development. To study the hormonal and developmental regulation of IGF-II gene expression, we have isolated a cDNA clone for rat IGF-II (rIGF-II) from a 12S [1.2-kilobase-pair (kbp)] fraction of mRNA from a rat liver cell line (BRL-3A) that directs the cell-free synthesis of pre-pro-rIGF-II. In the present study, the rIGF-II probe was used to determine the size of IGF-II RNA. Surprisingly, in BRL-3A cells and in neonatal liver, the probe hybridized under stringent conditions 10-20 times more strongly to a larger (4 kbp) RNA than to 1.2-kbp RNA. The 4-kbp RNA is almost exclusively cytoplasmic and is colinear with a 551-base fragment of the rIGF-II cDNA insert containing coding and 3' noncoding regions. The 4-kbp and 1.2-kbp RNA species are regulated coordinately with developmental age, being high in liver from neonatal rats but not detectable in liver from older animals, suggesting that both IGF-II mRNA species arise from a single primary transcript by alternative RNA processing. Although oligodeoxynucleotide hybridization and S1 nuclease protection experiments suggest that the 4-kbp RNA contains an intact protein-coding region, fractions enriched in 4-kbp RNA do not direct the translation of pre-pro-rIGF-II in vitro. This may indicate that the 4-kbp RNA specifies an altered protein product that has not yet been recognized, or alternatively that it contains a normal protein-coding region but requires further RNA processing to be activated for translation.