When 32P-labeled human neutrophils were activated by exposure to phorbol myristate acetate, three 48-kDa proteins (designated pp48/6.8, pp48/7.3, and pp48/7.8, from their isoelectric points) were found to have become labeled. With maximal stimulation, labeling was complete by 30 s. With lesser degrees of stimulation, the extent of labeling at 2 min correlated with rates of production by the phorbol-treated cells. Increased labeling of these 48-kDa proteins was also seen in cells exposed to f-Met-Leu-Phe. In phorbol-treated neutrophils from patients with X-linked cytochrome b558-negative chronic granulomatous disease, pp48/7.8 was labeled in a normal fashion, but pp48/6.8 and pp48/7.3 failed to take up 32P. In cells from patients with autosomal recessive cytochrome b558-positive chronic granulomatous disease, however, none of the three proteins took up 32P in response to phorbol. The three proteins appear to be very closely related, as indicated by the findings that phosphoserine was the only phosphoamino acid found in any of the three, and all three yielded identical one-dimensional phosphopeptide maps after digestion with either chymotrypsin or staphylococcal proteinase V8. These results reconcile earlier observations on protein phosphorylation in chronic granulomatous disease and provide further evidence for a relationship between the phosphorylation of this group of 48-kDa proteins and the activation of the respiratory burst oxidase.