Abstract The intracellular 66,000 D (66K) and 69,000 D (69K) matrix-like phosphoproteins of strain Colburn cytomegalovirus (CMV) have been compared with each other and with their electrophoretic counterparts in the virion. Three lines of evidence indicate that the 66K and 69K proteins are products of separate genes, and that the intracellular and virion :species are closely related. First, “pulse-chase” radiolabeling experiments showed that these proteins have separate precursors; that modification of each to the mature form correlated with phosphorylation; and that phosphorylation of the 69K precursor occurred more slowly than that of the 66K precursor, and resulted in a more dramatic slowing; of its electrophoretic mobility. Second, comparisons of the 66K and 69K proteins based on partial proteolysis of [ 35S]methionine-labeled proteins, using VS protease, and complete proteolysis of [ 32P]orthophosphate-labeled proteins, using trypsin or Pronase, provided no evidence of sequence relatedness. These analyses also suggested that the distribution of phosphorylated residues differs in the two proteins—clustered for the 69K and more disperse for the 66K. Phosphoamino acid analyses showed only phosphoserine in the 66K protein. The 69K protein contained, in addition to phosphoserine, an electrophoretically faster moving, unidentified spot. And third, immunological comparisons showed these proteins to exhibit little or no antigenic cross-reactivity. They did, however, demonstrate that the nuclear proteins are immunologically cross-reactive with their respective virion counterparts. Additional comparisons of these nuclear and virion proteins established that the virion 69K protein (v69) differs in electrophoretic mobility and net charge from the nuclear 69K protein but that it, as well as the virion 66K protein, has a two-dimensional phosphopeptide pattern similar to its nuclear counterpart.