To advance the serological diagnosis of coccidioidomycosis, a radioimmunoassay was developed to detect an antigen of Coccidioides immitis and evaluated with serum samples from patients and controls. The assay antigen was fractionated from a culture filtrate of C. immitis cells by Sephacryl S300 gel chromatography. The assay used preliminary acid-heat extraction to dissociate serum-bound antigen and staphylococcal protein A as an absorbent. Antigenemia was detected in five (56%) of nine patients with coccidioidomycosis. Three of four negative tests occurred with sera from patients who had already received antifungal chemotherapy. Antigenemia was not detected in 106 control patients and normal donors. One false-positive reaction was observed in a patient with histoplasmosis. In all nine patients with active coccidioidomycosis, anti-C. immitis antibodies were detected during the initial evaluation or in the follow-up. In summary, an immunoassay for C. immitis antigenemia was developed; antigenemia was detected in five (56%) of nine patients with active coccidioidomycosis and a positive test indicated active or persistent disease.