Acid phosphatase (AP) within the specific granules of eosinophils is held in a latent, inactive form prior to degranulation. Eosinophils responded to membrane stimuli with activation of AP throughout the granules of the cell, prior to degranulation. Activation occurred within the time required for fixation of the cells by glutaraldehyde. Whereas resting normal eosinophils demonstrated AP activity in 7.8 +/- 1.2 per cent of the granules, cells stimulated with opsonized staphylococci demonstrated activity in 78.9 +/- 5.0 per cent of the granules, and the amount of lead phosphate reactant deposited per active granule was considerably more intense in the stimulated than in the unstimulated cells. A similar stimulation of AP activity in eosinophils occurred with exposure to the surface-active agent phorbol myristate acetate or the calcium ionophore A23187. Activation was not altered by 5 microgram. per ml. of cytochalasin B or 4 x 10(-6) M colchicine. These aspects of the response closely parallel the stimulation of oxidative metabolism of eosinophils; however, the two were separable. Eosinophils obtained from a patient with chronic granulomatous disease, although unable to activate the normal oxidative metabolic burst, demonstrated a normal activation of AP.