Resident and thioglycollate-elicited macrophages maintained in culture for 24 h contain approximately 5 x 10(-16) and 12 x 10(-16) mol of ATP per cell, respectively. During particle ingestion, the levels of ATP in these cells did not change. However, the specific activity of ATP extracted from macrophages labeled with [32P]Pi during phagocytosis was 40% lower than ATP extracted from control cells. These results suggested that macrophages contain a high energy phosphate reservoir, in addition to the ATP pool(s). A search for such a reservoir led to the identification of creatine phosphate in both resident and thioglycollate-elicited macrophages at concentrations that are in 3- to 5-fold-molar excess over ATP. Creatine phosphate levels in phagocytosing resident macrophages decreased by 45%, while creatine phosphate levels in phagocytosing thioglycollate-elicited macrophages did not change. Creatine phosphate turnover was measured in macrophages prelabeled with [14C]creatine. Over 90% of the intracellular label was in the form of creatine phosphate. During phagocytosis, there was a 40% decrease in intracellular [14C]creatine phosphate in both resident and thioglycollate-elicited macrophages. These results indicate that creatine phosphate turns over more rapidly during phagocytosis and replenishes the ATP consumed.