A cDNA encoding the 37-kilodalton (kDa) capsid assembly protein of cytomegalovirus (CMV) strain Colburn was isolated from a lambda gt11 library constructed from CMV Colburn-infected human fibroblast RNA. RNA transcribed in vitro from this cDNA was translated in vitro to give a 40-kDa protein whose electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fragmentation pattern following partial proteolysis were indistinguishable from those of authentic assembly protein precursor. The position of the assembly protein gene was mapped to the EcoRI F, XbaI R, and SalI U restriction fragments, near the middle of the CMV Colburn genome, by Southern hybridizations using the cloned assembly protein cDNA as a probe. Similar sequences were identified by cross-hybridizations in colinear regions of the genomes of human CMV strains Towne and AD169: specifically, in the HindIII H, BamHI V, and EcoRI A fragments of Towne and in the HindIII L and BglII S fragments of AD169. The predominant transcript of the assembly protein gene was determined to be approximately 1 kilobase in size; however, a larger transcript (1.8 kilobases) was also identified. The nucleotide sequence of the assembly protein cDNA was determined and found to contain a single long open reading frame predicted to encode a polypeptide of 36.6 to 37 kDa, close to the 40-kDa size determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the assembly protein precursor. The presence of a single cysteine residue at the carboxy-terminal end of this open reading frame is consistent with data from biochemical studies and indicates that processing of the assembly protein precursor includes a proteolytic cleavage that removes its carboxyl end.