Several methods to increase the sensitivity of JC virus (JCV) DNA detection in cerebrospinal fluid (CSF) for a noninvasive diagnosis of AIDS-related progressive multifocal leukoencephalopathy (PML) were investigated. When CSF collected at clinical presentation was tested, JCV DNA was detected in 8 of 19 patients with PML by standard PCR (sensitivity, 42%; 95% confidence interval [CI], 21 to 66%) and in 14 of 19 by nested PCR (sensitivity, 74% [95% CI, 49 to 90%]; P = 0.014 [McNemar's test]. For multiple serial CSF samples, standard PCR yielded JCV DNA for 11 of 19 PML patients (sensitivity, 58% [95% CI, 34 to 79%]) and nested PCR yielded JCV DNA for 17 of 19 patients (sensitivity, 90% [95% CI, 66 to 98%]; P = 0.014). The majority of the false-negative samples were found to contain PCR inhibitors. Standard PCR did not detect JCV DNA in CSF from any of the 83 AIDS patients with other diagnosis (100% specificity [95% CI, 95 to 100%]); JCV DNA was found in CSF from one control patient by nested PCR (99% specificity [95% CI, 93 to 100%]).