The Bacillus subtilis cdd gene encoding cytidine/2'-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order trpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in lambda D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14,000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58,000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14,800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis sigma 43 and sigma 37 RNA polymerase holoenzymes during growth and stationary phase.