The winged helix transcription factor hepatocyte nuclear factor 3γ (HNF3γ) is expressed in embryonic endoderm and its derivatives liver, pancreas, stomach, and intestine, as well as in testis and ovary. We have generated mice carrying an Hnf3g-lacZ fusion which deletes most of the HNF3γ coding sequence as well as 5.5 kb of 3′ flanking region. Mice homozygous for the mutation are fertile, develop normally, and show no morphological defects. The mild phenotype change of the Hnf3g ^−/− mice can be explained in part by an upregulation of HNF3α and HNF3β in the liver of the mutant animals. Analysis of steady-state mRNA levels as well as transcription rates showed that levels of expression of several HNF3 target genes (phosphoenolpyruvate carboxykinase, transferrin, tyrosine aminotransferase) were reduced by 50 to 70%, indicating that HNF3γ is an important activator of these genes in vivo.