Previous studies have described three disulfide-bonded glycoprotein complexes within the envelope of human cytomegalovirus (HCMV). These have been designated gCI, gCII, and gCIII. Although gCI has been identified as homodimeric glycoprotein B (gB, gpUL55), the compositions of gCII and gCIII remain incompletely defined. Earlier studies suggested that gCIII was composed of glycoprotein H (gH, gpUL75) complexed with a second glycoprotein, the gL homolog of HCMV. We characterized the gCIII complex of HCMV using recombinant vaccinia virus-expressed gH and gL. Our results indicated that authentic gCIII was not reconstituted by coexpression of gH and gL. The presence of a third, structurally and antigenically unique glycoprotein with an estimated molecular mass of 125,000 Da in virion-derived gCIII complexes suggested that at least three proteins were necessary for formation of this envelope glycoprotein complex. This third glycoprotein, gp125, contained both simple and complex N-linked carbohydrates and had an estimated deglycosylated mass of 64,000 Da. Furthermore, we demonstrated that mature gH existed as both a covalently complexed and noncovalently associated component of the gCIII complex within the envelope of infectious extracellular virions. These findings provide further evidence for the structural complexity of the envelope of HCMV and emphasize the uncertainties associated with the previous assignment of specific functions to envelope proteins of HCMV.