Understanding the uses and limitations of a battery of laboratory methods is essential to diagnose histoplasmosis. Antigen detection and serology are valuable adjuncts to histopathology and culture. Improvements incorporated into the second-generation Histoplasma antigen assay have increased its sensitivity and specificity for diagnosis of histoplasmosis. More recently, the antigen assay has been modified to provide quantitation, which improves reproducibility and facilitates monitoring antigen clearance during treatment. Furthermore, detection of antigen in bronchoalveolar lavage fluid increases the sensitivity for diagnosis of pulmonary histoplasmosis. Serological tests for antibodies are also useful, but may be falsely negative in immunosuppressed patients. In addition, elevated antibody titres persist for several years following initial infection, complicating their interpretation. Although histopathology may provide for rapid diagnosis, its sensitivity is < 50% in patients with disseminated disease and even lower in pulmonary histoplasmosis. Polymerase chain reaction has been described, but sensitivity is less than that of histopathology. Culture, although highly specific, has notable limitations, including insensitivity, a need for invasive procedures and delayed growth. This review provides the background for understanding the role of a battery of diagnostic methods in histoplasmosis. Tests facilitating a rapid diagnosis are expected to improve the outcome in patients with severe disease.