Activation of 32P-loaded neutrophils with phorbol myristate acetate causes the labeling of a family of three 48K proteins that focus near neutral pH. The relationship between these phosphoproteins and the activation of the respiratory burst has been supported by the previous finding that phosphorylation was defective in the two most common types of chronic granulomatous disease (CGD): X-linked cytochrome-negative (X/-) and autosomal cytochrome-positive (A/+). In this report, these studies have now been extended to the rare A/- and X/+ forms of the disease. In all three patients with A/- CGD examined, the two most acidic 48K proteins failed to undergo enhanced phosphorylation in response to phorbol stimulation, a finding similar to that seen in X/- patients. In contrast, neutrophils from two patients with X/+ CGD appeared to phosphorylate the neutral 48K proteins in a normal fashion. It thus appears that the different phosphorylation patterns seen in chronic granulomatous disease are a reflection of the genetic heterogeneity of this disorder. These findings lend further support to the conclusion that the 48K phosphoprotein family is related to the respiratory burst, although not necessarily in a straightforward manner.