Reactivation of Toxoplasma gondii can lead to a life-threatening intracerebral infection in immunocompromised HIV-positive patients. Due to the current diagnostic limitations for establishing an exact diagnosis of cerebral toxoplasmosis, a nested PCR system was developed for direct detection of T. gondii in cerebrospinal fluid. A storage temperature for samples of -20 degrees C and sample preparation using Proteinase K appeared to be critical for obtaining a high sensitivity of PCR. A total of 56 samples from 38 HIV-positive patients and 12 HIV-negative patients with symptoms or signs of neurological disease were evaluated by PCR. 5 of the 38 HIV-positive patients were diagnosed as having cerebral toxoplasmosis and PCR was positive in samples from all 5 patients. In the remaining 33, PCR was positive in one case and negative in 32. An exact etiological diagnosis other than cerebral toxoplasmosis was established in 5 patients. PCR performed on cerebrospinal fluid samples seems to be a fast, sensitive, specific and valuable tool for establishing the diagnosis of cerebral toxoplasmosis among HIV-positive patients at the time of presentation of symptoms or signs of neurological disease.