The human cytomegalovirus (HCMV) UL98 gene is predicted to encode a homologue of the conserved herpesvirus alkaline nuclease. To determine if the HCMV UL98 gene product is functionally homologous to other herpesvirus alkaline nucleases, the HCMV UL98 protein was purified and its activity characterized in vitro. Extracts of HCMV-infected cells were fractionated using Q Sepharose, phosphocellulose and native DNA cellulose chromatography. UL98 immunoreactivity copurified with alkaline pH-dependent nuclease activity. The purified protein migrated at its predicted size of approximately 65 kDa in denaturing polyacrylamide gels, and displayed nuclease activity in an activity gel assay. Enzyme activity was characterized by a high pH optimum, an absolute requirement for divalent cation, salt sensitivity, and 5' to 3' exonuclease activity. DNA digestion resulted in 5' monophosphoryl mono- and oligodeoxyribonucleotides. Kinetic analyses revealed a turnover rate of greater than 200 per min, and similar apparent affinity and rate constants on single- and double-stranded DNA. These results indicate that a functional alkaline nuclease activity is conserved among distant members of the herpesvirus family, and are consistent with a highly conserved role in the virus life cycle.