The culture filtrate of Coccidioides immitis induced to replicate in its parasitic phase in vitro, termed endosporulation antigens (EA), was assayed for ability to stimulate human lymphocyte blastogenesis in vitro. Stimulation of lymphocytes from skin test-positive healthy subjects by EA was comparable to stimulation by spherulin at optimal dilutions, but EA are 500 times more potent when compared on the basis of weight. Both preparations slightly stimulated lymphocytes from skin test-negative subjects. Heating or dialysis of EA enhanced the effect on skin test-positive subjects, but concentration depressed it. Concentrated EA also depressed nonspecific stimulation caused by phytohemagglutinin. Dialysis of concentrated EA reduced the ability to depress responses. EA from an avirulent strain of C. immitis were as stimulatory as EA from a virulent strain, but concentrating the former did not produce as much depression as concentrating the latter did. A survey of subjects with an optimal dose of EA in lymphocyte transformation showed that EA could separate skin test-positive from -negative subjects as well as spherulin could. The survey also showed that a delta cpm (the difference of incorporated counts of tritiated thymidine per minute in the presence or absence of the reagent) of 10,000 is useful for this separation. These results also indicate the presence of suppressive substances in EA which are only partially dialyzable and which were significantly more prominent in a preparation from the virulent strain.