The rat HNF-3 (hepatocyte nuclear factor 3) gene family encodes three transcription factors known to be important in the regulation of gene expression in liver and lung. We have cloned and characterized the mouse genes and cDNAs for HNF-3 alpha, beta, and gamma and analyzed their expression patterns in various adult tissues and mouse embryonic stages. The HNF-3 proteins are highly conserved between mouse and rat, with the exception of the amino terminus of HNF-3 gamma, which in mouse is more similar to those of HNF-3 alpha and beta than to the amino termini of the rat HNF-3 gamma protein. The mouse HNF-3 genes are small and contain only two or three (HNF-3 beta) exons with conserved intron-exon boundaries. The proximal promoter of the mouse HNF-3 beta gene is remarkably similar to that of the previously cloned rat HNF-3 beta gene, but is different from the promoters of the HNF-3 alpha and gamma genes. The mRNA distribution of the mouse HNF-3 genes was analyzed by quantitative RNase protection with gene-specific probes. While HNF-3 alpha and beta are restricted mainly to endoderm-derived tissues (lung, liver, stomach, and small intestine), HNF-3 gamma is more extensively expressed, being present additionally in ovary, testis, heart, and adipose tissue, but missing from lung. Transcripts for HNF-3 beta and alpha are detected most abundantly in midgestation embryos (Day 9.5), while HNF-3 gamma expression peaks around Day 15.5 of gestation.