In contrast to quiescent cells, we currently report that purified cytomegalovirus (CMV), strain AD169, constitutively expresses phosphatidylserine (PS)-like procoagulant activity. Initial evidence for this came from the observation of a CMV-dependent decrease in factor Xa clotting times. In a purified system, the assembly of a functional complex between factor Xa and the cofactor Va to form prothrombinase was found to be dependent on the addition of CMV. The corresponding dense bodies (DB) and noninfectious enveloped particles had similar activity. Quantification of the total virion and DB phospholipid, and comparison of prothrombin conversion rates to experiments conducted using known concentrations of PS-containing vesicles showed that 8.5% and 7.2% of the CMV and DB phospholipid, respectively is procoagulant. Direct binding studies of 125I-labeled factor Xa, active site-blocked factor Xa, or the zymogen factor X, with virions or DB showed a single class of Ca(2+)-dependent sites with dissociation constants in the order of 10(-7) MOL/l. Immunogold electron microscopy confirmed the specificity of the CMV/factor Xa association. Cumulatively, these data suggest that the CMV surface contains the necessary procoagulant phospholipid for coagulation enzyme complex assembly. This may enable CMV (and possibly other enveloped viruses) to bypass an important physiologic regulatory mechanism for the production of thrombin.