DNase activity can be detected in lysates of extensively purified human cytomegalovirus (HCMV) particles. The presence of activity only in purified virions and not in dense bodies and its neutralization by HCMV-positive human sera strongly suggest that the enzyme is virus-specific. In situ analysis of the enzymatic activity of individual HCMV polypeptides, separated in a DNA-containing polyacrylamide gel, has shown that the DNase activity present in purified virions is mainly associated with a polypeptide of 46,000 dalton MW (p46). A monospecific anti-p46 serum, raised in mice, neutralizes about 80% of the DNase activity present in extracts of purified virions and recognizes an enzymatically inactive, 76,000 dalton MW polypeptide both in non-infected and HCMV-infected cells.