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The gene coding for the most abundant glycoprotein (gp58) of human cytomegalovirus (HCMV), strain AD169, was physically mapped on the viral genome. A monospecific rabbit antiserum against gp58 was used to screen a cDNA library that was constructed from poly(A)+ RNA of HCMV-infected cells in the prokaryotic expression vector λ gt11. A cDNA clone was identified which synthesized part of the glycoprotein. It allowed localization of the coding region within the right terminal sequence of the HindIII-F fragment between map coordinates 0·344 and 0·380 of HCMV virion DNA.