During the early phase of a human cytomegalovirus (HCMV) infection, the 2.7-kb early gene is by far the most abundantly transcribed RNA. Using strand-specific 32P or digoxigenin-labeled riboprobes derived from a subgenomic fragment of the HCMV Towne 2.7-kb early gene, we have performed Northern blot analysis and RNA in situ hybridization on human and mouse fibroblasts infected with HCMV and on 23 formalin-fixed paraffin-embedded sections of tissue obtained at autopsy. By Northern blot analysis, expression of the 2.7 kb early gene was found only in permissive infections. In contrast, specific hybridization was detected in both permissive and nonpermissive cells by RNA in situ hybridization. In nonpermissive cells, hybridization was weak and predominantly nuclear. In permissive cells, strong nuclear and cytoplasmic hybridization was noted. Specific hybridization to cells with and without cytopathic changes was detected with the anti-sense probe in CMV infected tissue obtained at autopsy. When the sense riboprobe was employed, no specific hybridization was found under nondenaturing conditions. These results suggest that in situ hybridization with a probe directed at the 2.7-kb early gene is an effective method of detecting both permissive and nonpermissive HCMV infections in different stages of infection and in localizing the expression of the major early gene in various cell types.