The C6 zinc cluster family of fungal regulatory proteins shares as DNA-binding motif the C6 zinc cluster, also known as the Zn(II)2Cys6 binuclear cluster. This family includes transcriptional activators like Gal4p, Leu3p, Hap1p, Put3p and Cha4p from Saccharomyces cerevisiae, qutA and amdR from Aspergillus, nit4 from Neurospora and Ntf1 from Schizosaccharomyces pombe. Seventy-nine proteins were retrieved from databases by homology to the C6 zinc cluster. All were fungal and 56 were found in the entire genome sequence of S.cerevisiae. Sequence analysis suggests that 60 of the 79 proteins possess one or more coiled-coil dimerization regions succeeding the C6 zinc cluster. Previous comparisons of Gal4p and seven other C6 zinc cluster proteins identified an additional region with weak homology. This region, designated the middle homology region (MHR), was shown to be present in 50 of the 79 proteins. Although reported mutation and deletion analyses suggest a role of MHR in regulation of protein activity, no function has yet been assigned specifically to this region. We find that the family of MHR sequences is confined to C6 zinc cluster proteins and hypothesize that one MHR function is to assist the C6 zinc cluster in DNA target discrimination.