The polC gene specifying DNA polymerase III (PolIII) of Staphylococcus aureus (Sa), was cloned with a novel strategy and found to contain a 4305-bp open reading frame (ORF) encoding a polypeptide of approx. 162 kDa. The 1435-codon ORF was engineered into an Escherichia coli (Ec) expression plasmid under the control of the lac promoter and its repressor. Derepression of Ec transformants carrying the recombinant (re-) vector generated high-level synthesis of active re-Sa PolIII. The re-PolIII was purified to > 98% homogeneity and was shown by N-terminal amino acid sequence analysis to be the bona fide product of the Sa polC ORF. The physical and catalytic properties of re-Sa PolIII and its responsiveness to inhibitors of the HPUra type were generally similar to those of Bacillus subtilis (Bs) PolIII. Comparative analysis of the primary structures of Sa PolIII, Bs PolIII and Mycoplasma pulmonis PolIII indicated strong conservation of essential catalytic domains and a novel zinc-finger motif. Comparison of the primary structures of Ec PolIII and these three Gram+ enzymes revealed a region of novel homology and reinforced the likelihood of a specific evolutionary relationship between PolIII of Gram+ and Gram- eubacteria. The polC gene mapped between omega 1074 [Tn551] and recA/ngr on the Sa NCTC 8325 genome.