Disseminated histoplasmosis is an unusual opportunistic infection in patients with advanced HIV infection living outside endemic areas. Diagnosis usually is made on the basis of isolation of Histoplasma capsulatum from clinical specimens or histologic examination. Reported is the case of an HIV-infected Columbian individual in whom the diagnosis of histoplasmosis was established within 24 h of collection of an adequate bronchoalveolar lavage specimen. The diagnosis was made by detection of specific fungal DNA and confirmed by isolation of Histoplasma capsulatum from blood, bone marrow and respiratory specimens 10 days later. The patient recovered under antifungal treatment and remained asymptomatic up to the last follow-up visit 6 months later. The polymerase chain reaction assay might be a powerful and rapid diagnostic tool for the diagnosis of non-European invasive fungal infections and should be further evaluated.