Rat liver insulin-like growth factor 1 (IGF-I) mRNAs encoding the same IGF-I and precursor exist as two predominant size classes of 7.5-7.0 and 1.2-0.9 kilobases (kb). The different sized mRNAs, differ only in length of 3' untranslated sequence (3'UT) and appear to derive from the use of alternate polyadenylation sites. Since the long 3'UT of the 7.5-7.0 kb IGF-I mRNAs contain sequences implicated in mRNA destabilization, we compared decay of the 7.5-7.0 and 1.2-0.9 kb mRNAs, during in vitro incubation in a reticulocyte lysate cell-free translation system. The 7.5-7.0 kb mRNAs decayed to 30% of the initial abundance after 10 min in reticulocyte lysate and were undetectable after 60 min. In contrast, 80% of the 1.2-0.9 kb mRNAs remained after 60 min in reticulocyte lysate. The rapid decay of the large IGF-I mRNAs during incubation in reticulocyte lysate demonstrates that analysis of mRNA translation in cell-free systems must take into account the rate of mRNA decay. Induction and decay of the two rat liver IGF-I mRNAs was also analyzed in vivo in hypophysectomized rats given a single i.p. injection of human growth hormone (GH). While GH induced parallel increases in abundance of the two mRNAs, their decay rates differed. The time taken for decay to 50% maximum abundance was 4 h for the 7.5-7.0 kb mRNAs and 14 h for the 1.2-0.9 kb mRNAs. Differential in vitro and in vivo half-lives of liver IGF-I mRNAs that encode the same protein, derive from the same gene, and differ only in length of 3'UT indicates novel post-transcriptional mechanisms for control of liver IGF-I synthesis. Such mechanisms may have major significance in non-hepatic tissues such as brain where the 7.5-7.0 kb IGF-I mRNAs predominate. These findings with IGF-I mRNAs have general implications for products of other genes with multiple polyadenylation sites.