Histoplasmosis is an infectious disease caused by inhaling spores of the fungal pathogen H. capsulatum and in Japan is considered an imported mycosis. However, some patients in Japan with histoplasmosis have no history of traveling overseas nor of risk of occupational exposure to Histoplasma. To investigate the possibility of native distribution of Histoplasma in Japan, 187 bat guano samples from 67 bat-inhabited caves in 17 prefectures were collected. These were examined for H. capsulatum by culture and Histoplasma-specific PCR in three independent laboratories. No H. capsulatum was detected by either method, therefore H. capsulatum is unlikely to be present in bat guano in Japanese caves.