We have determined the nucleotide sequence of a 2039-base pair segment of Escherichia coli chromosomal DNA containing the phr gene, which encodes deoxyribopyrimidine photolyase. The coding region of phr is 1416 base pairs and is preceded by regions homologous to consensus sequences for E. coli promoters and ribosome binding sites. The phr gene is preceeded by an open reading frame of 169 codons (orf169) which is transcribed in the same direction. The proximity of orf169 to phr suggests that both are members of a single operon containing one or more internal promoters allowing differential expression of phr. An unusually large number of rare or infrequently used codons are utilized in phr, which may contribute to the low copy number of photolyase. The sequence at the NH2 and COOH termini and the overall amino acid composition of mature photolyase, determined using purified protein, agrees with predictions based upon the nucleotide sequence. Photolyase consists of 471 amino acids and has a calculated molecular weight of 53,994.