In almost all cell types, adenylate cyclase is located in the plasma membrane. In lymphocytes, however, this enzyme has been claimed to be largely present in intracellular compartments. In this study, the distribution of adenylate cyclase activity in subcellular fractions of calf thymocytes was reinvestigated by a balance sheet approach. When subcellular fractionation was performed in the absence of ATP and dithiothreitol, less than a half of the homogenate basal activity could be recovered in the fractions, and this amount was distributed almost equally in three main compartments: the plasma membrane fraction, the microsomal and mitochondrial fractions and the nuclear fraction. However, if enzyme activity in the above fractions was measured in the presence of the stimulatory agents NaF, guanylylimidophosphate or guanosine 5'-O-(3-thio)triphosphate, or if the subcellular fractionation was performed in media containing ATP and dithiothreitol, the overall recovered activity greatly increased (up to 90%) and the distribution was shifted in favour of the plasma membrane fraction (up to 65% of the recovered activity). The adenylate cyclase properties were similar in all fractions. The ionophore alamethicin did not alter the subcellular distribution of the enzyme. The localization of adenylate cyclase in thymocytes thus appears to be primarily, if not uniquely, in the plasma membrane, as generally found in other cell types.