The hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is an autosomal dominant disorder that is due to mutations of the dual zinc-finger transcription factor, GATA3. The C-terminal zinc finger (ZF2) is crucial for DNA binding, whilst the N-terminal finger (ZF1) stabilizes the DNA binding by ZF2 and interacts with other multi-type zinc finger proteins, referred to as Friends of GATA (FOG), which modulate GATA3 function. The majority of GATA3 mutations causing HDR result in a disruption of ZF2 or its flanking region of basic amino acids, and these lead to a loss of DNA binding as shown by electrophoretic mobility shift assays (EMSAs). However, the manner in which one GATA3 missense mutation (Trp275Arg), which lies in ZF1 and does not affect ZF2, causes HDR has not been elucidated. We have therefore investigated this HDR mutation and another engineered ZF1 mutation (Cys264Arg) by EMSAs, dissociation assays, yeast two-hybrid assays and coimmunoprecipitation analysis. Both the Trp275Arg and Cys264Arg ZF1 mutants were shown to have normal DNA binding to a palindromic GATA site, but dissociation assays revealed that the stability of this binding was markedly reduced by the Cys264Arg mutation but not the Trp275Arg mutation. Yeast two-hybrid assays, aimed at studying the interactions between the wild-type and mutant GATA3 ZF1s and four of the eight zinc fingers of FOG2, revealed that wild-type GATA3 ZF1 interacted with FOG2 zinc fingers 1, 5, 6 and 8, whereas the Trp275Arg mutant interacts solely with FOG2 zinc finger 6, and the Cys264Arg mutant with none. These results were confirmed by co-immunoprecipitation analysis. Thus, our results demonstrate a novel mechanism whereby GATA3 mutations of ZF1 disrupt protein-protein interactions with FOG2 and lead to the developmental abnormalities of the parathyroids, inner ear and kidneys that occur in the HDR syndrome.