The polymerase chain reaction (PCR) was used to detect the presence of Paracoccidioides brasiliensis in a murine model of disseminated paracoccidioidomycosis. Using a previously identified P. brasiliensis-specific DNA sequence, P. brasiliensis DNA was detected in serum of five experimentally infected mice. The PCR method was able to detect as little as 10 pg of P. brasiliensis DNA in serum, and it was more sensitive than blood culture isolation (five of five were PCR positive versus two of five blood culture positive). There were no amplified fragments in serum from three noninfected control mice. Lung colony counts were similar in all infected mice and reflected a similar degree of P. brasiliensis infection at the time the samples were drawn. The relatively short processing time for the PCR, when compared with culture, its sensitivity, and the possibility of using serum samples for analysis, are important factors favoring this method for the diagnosis of paracoccidioidomycosis. Future studies should include the detection of P. brasiliensis in patients with different clinical forms of paracoccidioidomycosis.