Energy metabolism in Dictyostelium discoideum was studied by following the incorporation of [(3)H]adenine and [(32)P]Pi into the intracellular adenine nucleotides at two stages of differentiation. That the levels of nucleotides measured represented levels present in vivo was shown by demonstrating that the concentrations determined from cells quickly frozen in liquid nitrogen were identical to those from cells harvested and subsequently killed in perchloric acid. In addition, significant compartmentalization among the nucleoside triphosphates was not observed after 30 min, since uridine triphosphate, guanosine triphosphate, and adenosine triphosphate (ATP) were at radioactive equilibrium after exposure to [(32)P]Pi. At the sorocarp stage of differentiation, the dominant role of adenylate kinase activity is indicated by the observations that (i) adenylate kinase activity was found to be present, (ii) the specific radioactivities of the beta- and gamma-phosphates of ATP were equal, (iii) the increase in specific radioactivity of adenosine diphosphate was one-half that of ATP in the presence of [(32)P]Pi, and (iv) the steady-state equilibrium constant calculated (0.69) from the nucleotide levels equaled that of the apparent equilibrium constant reported in the literature (0.70). By using the same criteria, adenylate kinase activity was not the predominant reaction establishing the adenine nucleotide levels in cells at the pseudoplasmodium stage of differentiation.