This report describes the diagnosis of progressive multifocal leukoencephalopathy (PML) in nine patients using cytopathologic and histopathologic examination of computed tomographically guided stereotactic brain biopsies in combination with immunostaining for SV-40-related antigen and the polymerase chain reaction (PCR) for the JC virus. In four patients the diagnosis of PML was based on the microscopic appearance of the biopsies and immunostaining for SV-40-related antigen. In one of these patients the diagnosis was also supported by PCR for the JC virus. In two patients whose biopsies were only suggestive of PML, a definitive diagnosis was possible utilizing immunohistochemistry and PCR. In another case the histopathologic features were atypical of PML, and the diagnosis was established with immunostaining and PCR. The diagnosis of PML was established by PCR alone in two patients whose biopsies showed only suggestive or nonspecific findings. We conclude that the accuracy of stereotactic biopsy in the diagnosis of PML is enhanced by using a combination of light microscopy, immunohistochemistry and PCR.