As a result of multiple transcription initiation sites and differential splicing involving the first exons and alternate exon use, many insulin-like growth factor I (IGF-I) mRNA species are produced. In the present study, we have assessed the effect of GH on transcription start site usage and splicing and have determined the apparent in vivo translatability of IGF-I mRNAs with different 5'-untranslated region (UTR) sequences by comparing their abundance in total and polysomal RNA fractions from control, hypophysectomized, and GH-treated hypophysectomized rats. Hypophysectomy decreased the level of all IGF-I mRNA species, but those initiated at start site 3 in exon 1 and the major start site in exon 2 were preferentially reduced. These same variants were preferentially increased by GH treatment. Under all conditions, exon 1 mRNAs with shorter 5'-UTR sequences were enriched in polysomal RNA at the expense of IGF-I mRNAs with long 5'-UTR sequences, in accordance with the scanning model of translation initiation. Exon 2-derived mRNAs, on the other hand, which have short 5'-UTR sequences, were not enriched on polysomes, suggesting that some aspect of the exon 2-derived 5'-UTR other than length influences translation in vivo. These results demonstrate that transcription within exon 1 and between exons 1 and 2 is differentially regulated by GH status and that the variant IGF-I mRNA species resulting from the complex patterns of transcription initiation and splicing in these leader exons are differentially translated in vivo.