The present study was designed to investigate the prevalence of herpes simplex virus type-2 (HSV-2) in Egyptian patients with bladder cancer or cystitis and to evaluate the performance of different diagnostic HSV-2 assays. The study included 50 patients: 27 with bladder cancer (group I), 23 with cystitis (group II) and 20 subjects as controls (group III). HSV-2 DNA was detected using polymerase chain reaction (PCR) on bladder tissue and buffy coat cells (BCC). Electron microscopic studies (EMS) on BCC and ELISAs for IgM, IgG and specific glycoprotein G-2 (gG-2) IgG were performed. HSV-2 DNA was detected by PCR on bladder tissue biopsies in 29.6% and 21.7% of group I and II respectively and it was also detected by PCR on BCC in 22.2% and 21.7% of group I and II respectively. EMS revealed HSV like particles in 16.6% of cases. IgG, specific gG-2 IgG and IgM were detected in 30%, 16% and 6% of cases respectively. The different assays were evaluated in relation to PCR on bladder tissue biopsies. The gG-2-based ELISA and EMS on BCC were found to be highly specific (97.3% and 100% respectively), with similar low sensitivity of approximately 54%. PCR on BCC was the most sensitive assay. The association of HSV-2 with bladder cancer is suggested especially in schistosomal patients.