GATA transcription factors are crucial regulators of cell-specific gene expression in many tissues including the gonads. Although clinical cases of reproductive dysfunction have yet to be formally linked to GATA gene mutations, they have begun to be reported in other systems. Heterozygous GATA4 mutations have been associated with cases of congenital heart defects. Little is known, however, about the effect of these mutations on gonadal gene transcription. Since individuals carrying these mutations do not appear to suffer from gross reproductive defects, we hypothesized that this might be due to the differential transcriptional properties of the mutant proteins on heart versus gonadal target genes. Five mutations (S52F, E215D, G295S, V266M, and E359X) were recreated in the rat GATA4 protein. Several parameters were used to analyze the transcriptional properties of the mutants: activation of known gonadal target promoters (Star, Cyp19a1, and Inha), DNA binding, and interaction with GATA4 transcriptional partners. Three mutations (S52F, G295S, and E359X) reduced GATA4 transcriptional activity on the different gonadal promoters. With the exception of the G295S mutant, which showed a significant loss of DNA-binding affinity, the decrease in activity of the other GATA4 mutants was not associated with a change in DNA binding. All GATA4 mutants retained their ability to interact and cooperate with their major gonadal partners (NR5A1 and NR5A2) thereby compensating in part for the loss in intrinsic GATA4 transcriptional activity. Thus, unlike the heart, where the GATA4 mutations have deleterious effects, our data suggest that they would have a lesser impact on gonadal gene transcription and function.