Abstract 1. The properties of polynucleotide ligase from mouse cells infected with polyoma virus, and from uninfected mouse cells, were studied in order to investigate the possible involvement of ligase in polyoma DNA replication. 2. Two procedures for the assay of polynucleotide ligase were used to detect ligase in extracts of mouse embryo cells. Infection of the cells by polyoma virus resulted in a 2-fold stimulation of the ligase activity. On subcellular fractionation of both normal and polyoma-infected cells, 60 % of the ligase activity was in the isolated nuclei. The nuclear ligase was in two fractions, one soluble and the other insoluble. 3. The soluble ligases of polyoma-infected and normal mouse cells were purified 200-fold. The enzymes from infected and uninfected cells showed similar activities in the two assays; both enzymes had a molecular weight of about 220 000, showed a pH optimum between 7.2 and 7.8, and required Mg2+ and ATP ( K m = 1.5 · 10 −6 M ) for activity. 4. Temperature-sensitive mutants of polyoma were used in experiments to determine whether a new or an altered ligase appears in infected cells. None of the mutants tested affected the virus-induced stimulation of ligase activity. The question of whether the ligase which functions in the replication of polyoma DNA is specified by the viral or the host cell genome is discussed.