Tissue distribution and potential alternative splicing of insulin-like growth factor I (IGF-I) messenger RNA were studied using reverse transcriptase-polymerase chain reaction (RT-PCR) on RNA from several tissues at various stages of the life cycle of coho salmon (Oncorhynchus kisutch). DNA sequence analysis of RT-PCR products revealed three IGF-I mRNA transcripts, designated Ea-1, Ea-2, and Ea-3, which code for three distinct prohormones, IGF-IA-1, IGF-IA-2, and IGF-IA-3, respectively. The E-domain of proIGF-IA-1 is 35 amino acids long and shares 77% sequence identity with the E-domain of human proIGF-IA, which is also 35 amino acids long. The proIGF-IA-2 and proIGF-IA-3 E-domains are homologous to the proIGF-IA-1 E-domain but contain 27 and 39 amino acid inserts, respectively, between Lys86 and Glu87. In the human IGF-I gene Lys86 is coded by exon 4 and Glu87 is coded by exon 6. This suggests that Ea-2 and Ea-3 transcripts may be the result of alternative splicing during pre-mRNA processing. All three transcripts were readily detectable using a solution hybridization/RNase protection assay. Furthermore, RT-PCR and DNA sequencing analysis indicate the presence of three IGF-I prohormones in another member of the Salmonidae family, the Atlantic salmon (Salmo salar). An analysis of IGF-I and -II E-domains from several vertebrates suggests that certain chemical and physical properties of the molecule are well conserved despite wide variations in primary structure. Ea-1, Ea-2, and Ea-3 transcripts were found in whole embryos, and liver, muscle, and brain of juvenile and adult salmon. At least one IGF-I transcript was found in heart, kidney, testes, ovary, adipose tissue, and spleen of juvenile salmon. These results indicate that IGF-I is expressed during embryonic development of fish, and that most tissues are capable of IGF-I mRNA production. These data also indicate that pre-mRNA transcripts can be alternatively spliced to yield at least three prohormones.