Cytomegalovirus was assayed by in situ hybridization in 29 consecutive specimens obtained by open-lung biopsy. Biotin-labeled cytomegaloviral DNA was hybridized to formalin-fixed, paraffin-embedded tissue sections and was detected by an immunoperoxidase procedure. The sensitivity of this technique was similar to that of both viral culture and viral antigen detection in frozen sections by monoclonal antibody. Cytomegaloviral infection was diagnosed within 24 hr by study of the formalin-fixed, paraffin-embedded sections of lung tissue. The assay was specific, reproducible, and readily applicable both to the study of multiple tissue sections for diagnostic purposes and to retrospective studies. Hybridization studies of multiple sections from two patients with fatal cases of pneumonia showed that cytomegaloviral infection is not homogeneous in all cases.