One hundred ninety-four bronchoalveolar specimens were evaluated by microscopic examination and by amplification of a sequence of a Pneumocystis carinii dihidropteroate synthase gene for identification of mutations linked to sulfa resistance. PCR sensitivity and specificity were 100 and 86.7%, respectively, compared to results of microscopic examination. However, 7 out of 19 microscopy-negative, PCR-positive samples were collected from subjects with a clinically high probability of P. carinii pneumonia, suggesting that PCR may be more sensitive than microscopic examination, although the absolute performance of PCR cannot be determined. Mutations were identified in 28 out of 70 (40%) PCR-positive specimens and were significantly more common in patients exposed to sulfa drugs (21 out of 29 [72.4%]) than in those not exposed to sulfa drugs (4 out of 35 [11.4%]).