Cytomegalovirus (CMV) proteins from isolates of both human (HCMV) and simian (SCMV) origin have been compared. Three classes were analyzed: the immediate-early (IE) proteins, other infected-cell-specific proteins not present in virus particles, and the proteins that constitute the mature extracellular virion. Comparisons were based on one- and two-dimensional (charge-size) separations in denaturing polyacrylamide gels, and on the selectivity of biosynthetic radiolabeling with [32P]orthophosphate and [3H]glucosamine. Results indicate that most, if not all, of the HCMV and SCMV proteins recognized, have counterparts in strain Colburn. As a group, the simian strains exhibit protein similarities that distinguish them from the human strains. Among the most diagnostic of these are the 205K and 145K virion proteins, each of which is about 7K smaller than its HCMV counterpart, and the predominant IE proteins, which are 10K to 20K (depending upon the strain) larger than their HCMV counterparts. The proteins of strain Colburn are shown to be more like those of the simian isolates than the human, and more like those of a vervet strain than rhesus. Leads provided by experiments using strain Colburn have aided in the identification of a previously unrecognized, abundant virion protein that is a principal phosphate acceptor, both in vivo and in vitro. Three additional phosphorylated proteins are identified in HCMV virions, as well as three glycoproteins. Only two HCMV strain-specific protein differences were detected by comparisons based on separation in SDS-containing polyacrylamide gels--one in the IE protein of strains Towne and Davis; the other in a virus capsid protein of strain AD169.