Pneumocystis causes pneumonia in immunodeficient hosts but also likely causes infection in healthy hosts. To characterize infection in healthy mice, we developed and validated a real-time polymerase chain reaction assay for quantitation of Pneumocystis carinii f. sp. muris. In healthy mice exposed to Pneumocystis-infected animals, organisms were first detected at 2-3 weeks, peaked at 5-6 weeks, and were cleared by 7-9 weeks. The peak organism load in healthy animals was 2-3 logs lower than that in immunodeficient animals. This approach should facilitate studies of anti-Pneumocystis immune mechanisms in healthy hosts and provide insights into the development of Pneumocystis pneumonia in immunodeficient hosts.