A concern currently exists regarding the potential for exposure of health care workers to pentamidine isethionate, a drug used for prevention and treatment of Pneumocystis carinii pneumonia in immunocompromised patients, including those with human immunodeficiency virus infection. In order to evaluate worker exposures, a sampling and analytical method for pentamidine isethionate in air has been developed. This method involves sampling with a 37-mm PVC membrane filter at 1 to 2 L/min, recovery with 3 mL of 50:50 ethanol:water with 0.085% phosphoric acid and 0.04% tetramethylammonium chloride in an ultrasonic bath for 10 min, and analysis by high performance liquid chromatography with fluorescence detection. The limit of detection is about 18 ng per sample, and the lower limit of quantitation is 50 ng per sample. Recoveries of pentamidine isethionate from PVC filters were 0.76 to 0.91 at 50 to 8820-ng levels of fortification. Samples were stable on PVC filters during 27 days of storage at room temperature. Because patients who are treated with pentamidine isethionate are at increased risk of contracting tuberculosis (TB), safety precautions for handling samples contaminated with TB were included in the sampling and analytical method.