The silk glands of Bombyx mori, a highly replicative tissue contains high levels of DNA polymerases alpha, delta and epsilon but not DNA polymerase-beta. However, we detected the latter activity in the gonadal tissues, viz. the pupal ovaries and testes of B. mori. The enzyme has been purified to homogeneity from the pupal ovaries by a series of column chromatographic and affinity purification steps. The enzyme satisfied the criteria to be designated as DNA polymerase-beta based on its small size, requirement for high concentration of monovalent cations for catalytic activity, sensitivity to ddTTP and insensitivity to aphidicolin. It is a monomeric polypeptide of M(r) 40 kDa, and the Km for dNTPs ranges between 8-20 microM. DNA polymerase-beta is biochemically and immunologically distinct from DNA polymerase-alpha from the silk glands of B. mori. The enzyme showed a preference for gapped DNA, and could not elongate ultraviolet irradiated template beyond the pyrimidine dimers. The absence of any associated primase and exonuclease activities from this enzyme, and its conspicuous absence in the highly replicative tissue, imply that it is unlikely to participate in the DNA endoreplication process.