The pancreatic regenerating (reg) gene is proposed to be involved in pancreatic beta-cell growth. Up- or down-regulation of reg gene expression has been shown to parallel variations in beta-cell mass and function in the adult pancreas. In several species at least two nonallelic reg genes have been identified. In this study we investigated the expression of each individual reg gene (reg-I and reg-II) during embryogenesis in the mouse. Single mouse embryos were harvested at 8.5, 9, 10, and 12 days of development, homogenized and subjected individually to reverse transcription (RT)-PCR, with a single primer pair to amplify both reg-I and -II mRNAs. Southern blot analysis of the RT-PCR products revealed the presence of reg mRNA at day 9 of embryogenesis, just before the beginning of pancreatic organogenesis. Slot-blot analysis with internal oligonucleotide probes that specifically recognize reg-I or -II sequences demonstrated that only reg-I mRNA was present in day 9 and day 10 prepancreatic embryos. Reg-II mRNA was not detected until day 12, a stage corresponding to late organogenesis. RT-PCR for insulin mRNA from the same samples used for the amplification of reg mRNA showed that the earliest insulin expression occurred at day 8.5, and coincided with the onset of reg-I expression. Hybridization with gene-specific oligonucleotide probes revealed that only insulin-II mRNA was detectable at this time. Insulin-I mRNA was not detectable until day 12 and coincided with early reg-II expression. These results suggest that the two nonallelic reg genes and the two insulin genes are expressed differentially during early embryogenesis. Differential expression of reg-I and -II suggests that they may be induced by different and independent stimuli and have distinct functions.