We developed a modified double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that detected relatively low concentrations of known Pneumocystis carinii antigen added to buffer or rat sera. Artificial immunization-derived polyclonal rabbit anti-P. carinii antibody was used on the solid phase to capture the antigen. Infection-derived (after P. carinii pneumonia) polyclonal rat anti-P. carinii antibody or a mixture of five murine monoclonal antibodies was used as the antigen detector antibody. Rabbit anti-rat immunoglobulin G antibody or goat anti-mouse immunoglobulin G antibody conjugated to alkaline phosphatase was used as the final antibody. After standardization and optimization of the various reactants in this ELISA system, approximately 53 ng of known P. carinii antigen per ml suspended in phosphate-buffered saline-Tween 20 buffer or 210 ng of antigen per ml suspended in normal rat serum diluted 1:4 could be detected. In addition, an indirect ELISA for P. carinii antibody measurement was developed, using as the antigen a soluble supernatant from a sonicated preparation of Percoll-purified whole cysts and trophozoites to coat the solid phase. Limited studies with sera from a small number of caesarian-obtained, barrier-sustained rats from Charles River Breeding Laboratories, Inc., and the National Institutes of Health and sera from normal and heavily infected rats indicated that the caesarian-obtained, barrier-sustained rats had negligible levels of antibody. The normal and heavily infected rats had variable antibody titers. A significantly high level of P. carinii antigenemia was detected in only 2 (11%) of 18 heavily infected rats. Extensive studies of the P. carinii pneumonia rat model with the ELISA did not reveal significant serum P. carinii antigenemia during the acute stage of infection. However, soluble P. carinii antigen was detected by the ELISA and Western blot assays in the supernatant of lavage fluid after centrifugation to sediment intact organisms. As expected, P. carinii antigens were detected by these assays in the lavage pellet recovered after centrifugation. In conclusion, the antigen assay used in this study detected P. carinii antigen in lung lavage but failed to detect P. carinii antigen in rat serum during the acute phase of infection.