The effect of a Coccidioides immitis spherule-derived antigen (spherulin) upon in vitro thymidine uptake by sensitized and nonsensitized lymphocytes was studied, and optimal conditions for the assay were determined. The mitogenic portion of the antigen was shown to be nondialyzable, as was true for a mycelial-derived antigen (coccidioidin). Concentrations of 20 mug of nondialyzable spherulin solids and 17 mug of nondialyzable coccidioidin solids per ml of culture were shown to be suitable for differentiating skin test-positive from skin test-negative individuals. Comparison with mycelial filtrate suggested spherulin may be a more potent mitogen for sensitized lymphocytes.