The differentiation of mediastinal masses caused by lymphoma from those caused by histoplasmosis may require thoracotomy. We reviewed the medical records of 37 children undergoing initial evaluation for anterior or middle mediastinal masses. Sixteen had biopsy-proved lymphoma, and 21 had histoplasmosis; seven with histoplasmosis underwent thoracotomy. Age, sex, fever, weight loss, duration of illness, anemia, erythrocyte sedimentation rate, nonspecific reactants, and lung infiltrates and calcifications were similar in both groups. Masses were in the middle mediastinum in all patients with histoplasmosis and in 69% with lymphoma. Masses were in the anterior mediastinum in one of 21 (5%) with histoplasmosis and 13 of 16 (81%) with lymphoma. Among patients with lymphoma, histoplasmal complement fixation antibody titers were less than 1:8 in 14 of 15 (93%); a single patient had a titer of 1:16. The CF titers were greater than or equal to 1:32 in 14 of 21 (67%) with histoplasmosis. In children with middle mediastinal masses, a histoplasmal CF yeast or mycelial titer greater than or equal to 1:32 is strongly suggestive of acute histoplasmosis and biopsy is not required. Children not fulfilling these criteria should undergo diagnostic biopsy.