A skin test-active fraction was isolated from the mycelial-phase cell walls of Coccidioides immitis. This alkali-soluble, water-soluble antigen (C-ASWS) elicited positive reactions in 22 of 24 (92%) of the Coccidioides-sensitized guinea pigs whereas only 14 (54%) of the same guinea pigs reacted to commercial coccidioidin (BioCox). None of the 21 Histoplasma-sensitized guinea pigs cross-reacted with the C-ASWS antigen. Footpad tests in mice actively infected with Coccidioides further established the efficacy of the C-ASWS antigen in eliciting a delayed-type hypersensitivity response. One-microgram doses of C-ASWS produced reactions comparable to 100-mug doses of nondialyzable coccidioidin (Smith's lot 64 D4). The C-ASWS fractions isolated from three different C. immitis strains showed similar reactivity in terms of the number of positive reactions produced in Coccidioides-sensitized guinea pigs. However, the induration responses (diameter in millimeters) elicited by the C-ASWS fraction of one strain were significantly less than those elicited by the C-ASWS fractions of the other two C. immitis strains.