The diagnosis of definite progressive multifocal leukoencephalopathy (PML) has been a neuropathological domain. We reviewed all Human Immunodeficiency Virus Type 1 (HIV-1) seropositive patients in our institution between 01.01.1989 and 31.12.1994 and identified 20/823 cases with PML by clinical and imaging criteria. Diagnosis was neuropathologically confirmed in 5 cases. Diagnostic criteria included rapid onset (< 2 weeks) of multifocal neurological signs and symptoms, advanced immunosuppression and asymmetric uni- or multifocal white matter lesions without mass effect, contrast enhancement or cortical atrophy in magnetic resonance imaging (MRI). The overall incidence of PML was stable over the observation period (approximately equal to 2.5%). The mean age at onset (41.7 years) was significantly lower compared to HIV-1 seronegative PML patients (peak in the sixth decade of life), male patients prevailed (100%). Mean survival (3.9 months) was extremely short. Human polyoma virus JC (JCV) polymerase chain reaction (PCR) in the cerebrospinal fluid (CSF) demonstrated a considerable rate of possible cerebral co-infection with HIV-1 and JCV as well as subclinical infection with JCV. Therefore demonstration of JCV deoxyribonucleic acid by PCR in the CSF alone is not sufficient for clinical PML diagnosis. We present diagnostic criteria on the basis of epidemiological, neuroradiological and CSF parameters that allow us to make the clinical diagnosis of PML. Although quick and safe, routine stereotactic brain biopsy is not necessary to confirm the diagnosis.