The means by which humans acquire Pneumocystis carinii is not well understood. Whether it can be acquired from specific environmental sources or transmitted from person to person has not been determined. This study was designed to detect nucleic acids of P. carinii in air samples from various locations, including P. carinii-infected patients' homes and hospital rooms, non-P. carinii-infected patients' hospital rooms, empty hospital rooms, offices at Indiana University, and other homes in different locations. DNA was extracted from cellulose-ester filters through which air samples had been filtered, and the P. carinii DNA was amplified by PCR with primers specific for the internal transcribed spacer regions of rRNA. P. carinii DNA was found in 17 of 30 air samples (57%) from the rooms of P. carinii-infected patients. It was also found in 6 of the 21 other hospital rooms sampled (29%) but was not found in any of the offices, storage areas, or control homes. Environmental sampling suggests that the airborne presence of P. carinii genetic material and infectious organisms is plausible. The organism was also detected in locations where P. carinii patients were not immediately proximate, such as the hospital rooms of non-P. carinii-infected patients.