The Saccharomyces cerevisiae CDC43 gene product is involved in establishing cell polarity during the cell-division cycle. When grown at restrictive temperatures, temperature-sensitive cdc43 mutants are unable to form buds and display delocalized cell-surface deposition [Adams et al., J. Cell Biol. (1990) in press]. We have isolated a cdc43-complementing plasmid from a yeast genomic-DNA library and localized the CDC43 gene, by subcloning and transposon-mutagenesis experiments, to a 1.2-kb region of DNA that contained only one significant ATG-initiated open reading frame of 213 codons. The putative CDC43 gene product contains a possible nuclear-localization signal sequence, a cysteine-rich domain and a histidine-rich domain, and a region that is similar in structure to alpha-helix-turn-alpha-helix structural domains present in some prokaryotic and eukaryotic DNA-binding proteins.