Human cytomegalovirus (HCMV) DNA was digested with restriction endonucleases and the fragments characterized with respect to molecular weight and relative mole proportions. The terminal fragments were identified by digesting HCMV DNA with exonucleases before restriction endonuclease treatment and subsequent gel analysis. The HindIII fragments of HCMV DNA were cloned in Escherichia coli and recombinant plasmids were characterized by digestion with restriction endonucleases and by molecular hybridization with HindIII, Bg/II and XbaI fragments of the virus genome. Data from these experiments were used to construct physical maps of HCMV DNA for the HindIII, Bg/II and XbaI restriction endonucleases. The terminal regions of the genome and the region containing fragment HindIII M were shown to be heterogeneous.