Transcription factor GATA4 is required for the development and function of the mammalian gonads. We first reported that the GATA4 gene in both human and rodents is expressed as two major alternative transcripts that differ solely in their first untranslated exon (exon 1a vs. exon 1b). We had also showed by quantitative PCR that in mouse tissues, both Gata4 exon 1a- and 1b-containing transcripts are present in all sites that are normally positive for GATA4 protein. In adult tissues, exon 1a-containing transcripts generally predominate. A notable exception, however, is the testis where the Gata4 exon 1a and 1b transcripts exhibit a similar level of expression. We now confirm by in situ hybridization analysis that each transcript is also strongly expressed during gonad differentiation in both sexes in the rat. To gain further insights into how Gata4 gene expression is controlled, we characterized the mouse Gata4 promoter sequence located upstream of exon 1b. In vitro studies revealed that the Gata4 1b promoter is less active than the 1a promoter in several gonadal cell lines tested. Whereas we have previously shown that endogenous Gata4 transcription driven by the 1a promoter is dependent on a proximally located Ebox motif, we now show using complementary in vitro and in vivo approaches that Gata4 promoter 1b-directed expression is regulated by GATA4 itself. Thus, Gata4 transcription in the gonads and other tissues is ensured by distinct promoters that are regulated differentially and independently.