The cytoplasmic pH undergoes a biphasic change when neutrophils are activated. The role of Ca 2+ in initiating these changes was investigated. No correlation was found between the increased cytosolic [Ca 2+ ] and the stimulation of the Na + /H + antiport. Similarly, the cytoplasmic acidification elicited by activation in Na + -free media was found to be unrelated to [Ca 2+ ]. Reversal of Na + /H + exchange was also ruled out as the source of the acidification. Data using a variety of soluble activators indicate that metabolic acid generation is largely responsible for the observed drop in cytoplasmic pH.