Genes encoding the chi and psi accessory proteins of the DNA polymerase III holoenzyme replicase of Escherichia coli have been identified, sequenced, and used to express and purify both chi and psi in quantity. The holC gene encoding chi is located between the xerB and valS genes at 96.5 min on the chromosome; it encodes a 147-amino acid protein of 16.6 kDa. holD encoding psi lies upstream of rimI at 99.3 min and encodes a 137-amino acid protein of 15.2 kDa. The genes have been cloned into expression vectors, and both chi and psi have been purified in quantity. The accompanying report characterizes the function and physical interactions of chi and psi with other holenzyme subunits (Xiao, H., Dong, Z., and O'Donnell, M. (1993) J. Biol. Chem. 268, 11779-11784).