Three types of virus particles have been recovered from the culture medium of human foreskin fibroblasts infected with human strains of cytomegalovirus (HCMV). Two of these, virions and dense bodies, are routinely observed and have been described by others. The third, produced in lesser amounts, has not been previously characterized. This particle, separable from virions by rate-velocity sedimentation, is morphologically distinguished from them only by core structure. Radiolabeling and biological assays have established that these particles, like dense bodies, lack DNA and are not infectious. Based on these properties, we have designated this virion-like structure as a noninfectious enveloped particle (NIEP). Comparisons of the protein constituents of these three particles has shown that dense bodies have the simplest composition. Approximately 95% of their protein mass is represented by a 69,000 Da (69K) matrix-like protein. While dense bodies appear to have a normal complement of virion glycoproteins, they completely lack other predominant virion species. The protein compositions of virions and NIEPs are more complex than that of dense bodies, and are distinguished from one another by the presence in NIEPs of a 35,000 Da (35K) protein absent from the two other particles. Biosynthetic radiolabeling and cell fractionation experiments have demonstrated that this 35K protein is produced only in infected cells, is phosphorylated and partitions with the nuclear fraction. These and other results suggest that this protein is the HCMV counterpart of the previously described B-capsid proteins VP22a of herpes simplex and 37K of CMV (strain Colburn). NIEPs are produced by all HCMV strains examined and have not been observed in preparations of herpes simplex virus- or Old World monkey CMV-infected cells. Although this particle is generally present in much lower amounts than virions, strain AD169 overproduces NIEPs by approximately 10-fold. We have also found that the additional NIEP protein of AD169 has an apparently larger size (i.e., 36K) than the corresponding protein of other strains. The correlation between AD169 NIEP overproduction and its altered protein suggests that the two may be causally related.