The transcription factor hepatocyte nuclear factor 4 (HNF4) is an orphan member of the nuclear receptor superfamily expressed in mammals in liver, kidney, and the digestive tract. Recently, we isolated the Xenopus homolog of mammalian HNF4 and revealed that it is not only a tissue-specific transcription factor but also a maternal component of the Xenopus egg and distributed within an animal-to-vegetal gradient. We speculate that this gradient cooperates with the vegetally localized embryonic induction factor activin A to activate expression of HNF1alpha, a tissue-specific transcription factor with an expression pattern overlapping that of HNF4. We have now identified a second Xenopus HNF4 gene, which is more distantly related to mammalian HNF4 than the previously isolated gene. This new gene was named HNF4beta to distinguish it from the known HNF4 gene, which is now called HNF4alpha. By reverse transcription-PCR, we detected within the 5' untranslated region of HNF4beta two splice variants (HNF4beta2 and HNF4beta3) with additional exons, which seem to affect RNA stability. HNF4beta is a functional transcription factor acting sequence specifically on HNF4 binding sites known for HNF4alpha, but it seems to have a lower DNA binding activity and is a weaker transactivator than the alpha isoform. Furthermore, the two factors differ with respect to tissue distribution in adult frogs: whereas HNF4alpha is expressed in liver and kidney, HNF4beta is expressed in addition in stomach, intestine, lung, ovary, and testis. Both factors are maternal proteins and present at constant levels throughout embryogenesis. However, using reverse transcription-PCR, we found the RNA levels to change substantially: whereas HNF4alpha is expressed early during oogenesis and is absent in the egg, HNF4beta is first detected in the latest stage of oogenesis, and transcripts are present in the egg and early cleavage stages. Furthermore, zygotic HNF4alpha transcripts appear in early gastrula and accumulate during further embryogenesis, whereas HNF4beta mRNA transiently appears during gastrulation before it accumulates again at the tail bud stage. All of these distinct characteristics of the newly identified HNF4 protein imply that the alpha and beta isoform have different functions in development and in adult tissues.