The association between human cytomegalovirus (HCMV) infection and glioblastoma has been a source of debate in recent years because of conflicting laboratory reports concerning the presence of the virus in glioma tissue. HCMV is a ubiquitous herpesvirus that exhibits tropism for glial cells and has been shown to transform cells in vitro. Using sensitive immunohistochemical and in situ hybridization methods in 50 glioma samples, we detected HCMV antigen and DNA in 21/21 cases of glioblastoma, 9/12 cases of anaplastic gliomas and 14/17 cases of low-grade gliomas. Reactivity against the HCMV IE1 antigen (72 kDa) exhibited histology-specific patterns with more nuclear staining for anaplastic and low-grade gliomas, while GBMs showed nuclear and cytoplasmic staining that likely occurs with latent infection. Using IHC, the number of HCMV-positive cells in GBMs was 79% compared to 48% in lower grade tumors. Non-tumor areas of the tissue contained only four and 1% of HCMV-positive cells for GBMs and lower grade tumors, respectively. Hybridization to HCMV DNA in infected cells corresponded to patterns of immunoreactivity. Our findings support previous reports of the presence of HCMV infection in glioma tissues and advocate optimization of laboratory methods for the detection of active HCMV infections. This will allow for detection of low-level latent infections that may play an important role in the initiation and/or promotion of malignant gliomas.