Structural gene mutants were cloned and exploited to identify the major catalytic domains of Bacillus subtilis DNA polymerase III (BsPolIII), a 162.4-kDa [1437 amino acids (aa)] polymerase: 3'-5' exonuclease (Exo) required for replicative DNA synthesis. Analysis of the sequence, mutagenicity, and catalytic behavior of natural and site-directed point mutants of BsPolIII unequivocally located the domain involved in exonuclease catalysis within a 155-aa residue segment displaying homology with the Exo domain of Escherichia coli DNA polymerase I. Sequence analysis of four structural gene mutations which specifically alter then enzyme's reactivity to the inhibitory dGTP analog, 6-(p-hydroxyphenylhydrazino)uracil, and the inhibitory arabinonucleotide, araCTP, defined a domain (Pol) involved in dNTP binding. The Pol domain was in the C-terminal fourth of the enzyme within a 98-aa segment spanning aa 1175-1273. The primary structure of the domain was unique, displaying no obvious conservation in any other DNA polymerase, including the distantly related PolIIIs of the Gram- organisms, E. coli and Salmonella typhimurium.