Highly active antiretroviral therapy (HAART) has decreased the incidence of opportunistic infections in the central nervous system (CNS) in AIDS patients. However, toxoplasmic encephalitis (TE) still represents the most common cerebral mass lesion in patients infected with human immunodeficiency virus (HIV). The aim of this study was to evaluate nested PCR-B1 using cerebrospinal fluid (CSF) to detect Toxoplasma gondii DNA for the diagnosis of TE. A total of 114 samples were evaluated, and 33/44 samples from patients with TE were positive by PCR (sensitivity 75%), demonstrating the diagnostic usefulness of PCR technique. PCR-B1 products were analyzed by restriction fragment length polymorphism (RFLP) in 30 samples. Only type I allele at B1 was identified in these samples according banding patterns. This is the first report of evaluation of S1-AS1/S2-AS2 set of primers in more than 100 clinical samples as well as the first genotyping study of T. gondii in Cuba. © 2009 Elsevier Inc. All rights reserved.