We used a bioassay to measure pentamidine concentrations in autopsy specimens from 22 patients with AIDS. Patients received pentamidine isethionate (approximately 4 mg/kg per day) parenterally for Pneumocystis carinii pneumonia; one received monthly prophylaxis. We found that lung levels of 30 micrograms/g were achieved only after the fifth dose; tissue accumulation was usually greater in the liver, kidney, adrenal, and spleen than in the lung; detectable levels were present in some tissues as late as one year after the last dose; and low but detectable levels were present in the brain of six of 17 patients. Two patients had no detectable lung levels after two days of therapy; one had a level of 17.5 micrograms/g after four doses, and two had levels of 30 micrograms/g after five doses. A more rapid and effective method of delivery, such as aerosol, should achieve higher concentrations earlier. Because pentamidine persists in lung tissue over days to weeks, daily administration may not be necessary.