In the mouse, insulin is produced from two similar but nonallelic genes that encode proinsulins I and II. We have investigated expression of these two genes during mouse embryonic development, using a PCR to detect the two gene transcripts and immunocytochemistry to visualize the two corresponding proteins. At appearance of the dorsal pancreatic anlage at day 9.5 of gestation, both mRNAs could be detected in the embryos, and both proteins were present together in the same cells of the developing pancreas. At days 9.5 and 10.5, when the ventral anlage appears, there were fewer proinsulin H mRNAs than proinsulin I mRNAs. At day 12.5 this ratio was reversed. Proinsulin II mRNA, but not proinsulin I mRNA, could be detected at day 8.5 in the prepancreatic embryo. Proinsulin II mRNA, but not proinsulin I mRNA, was also found in the heads of embryos at day 9.5 and at all later stages studied. These results indicate that the two proinsulin genes are regulated independently, at least in part. They also suggest that insulin might play a role as a growth factor in the developing mouse brain.Insulin, a key hormone in metabolic homeostasis, is synthesized, stored, and secreted by beta cells of the pancreatic islets. The protein is synthetized in the form of a precursor, preproinsulin, which is highly conserved among animal species. Unlike most mammals, mice and rats express two nonallelic genes that encode proinsulins I and II. In the mouse these two proteins differ by two amino acids in the B chain and three amino acids in the C peptide. Mouse C peptide I also lacks the Gly-Ala residues present in positions 17 and 18 of C peptide 11(1, 2). The corresponding genes in mouse and rat are highly homologous, and their organization is similar, except that the preproinsulin I gene possesses only the first of two introns present in the preproinsulin II gene (1, 3).On previous studies concerning the regulation of these two nonallelic genes, mRNAs were reported to be present in nearly equal quantities in adult pancreas of mouse (1, 4), as well as of rat (5) Here we have investigated regulation of the expression of the two proinsulin genes during mouse development. In the mouse, the pancreas is derived from the duodenum as two evaginations evolving at days 9.5 (dorsal) and 10.5 (ventral) of the gestation. The evaginations coalesce at day 11, and insulin has been first detected in previous studies at day 11.5 (9, 10). We have used a reverse-transcriptase-PCR (RT-PCR) assay, which allows identification and estimation of the relative amounts of mRNA transcribed from each of the two proinsulin genes. This RT-PCR, using a single pair ofprimers and a single probe for the two transcripts, allowed comparison of the relative amounts of proinsulin I and II mRNAs in the samples (11). Immunocytochemistry with antibodies specific for each of the two proinsulins allowed us to distinguish the products of the two genes in embryo sections. We have, thus, been able to detect insulin mRNAs and proteins much earlier, to distinguish the two forms, ...