The effect of exogenously added adenylate cyclase from Bordetella pertussis (strain 114) has been investigated in Y-1 mouse adrenal tumor, chinese hamster ovary (CHO) and several other cells. A partially purified adenylate cyclase was found not to enter cells but, nevertheless, produced large amounts of cAMP in the medium. We could show that this resulted from release of ATP (and not larger molecules). The ATP released by the cells could be (1) directly measured and was replenished after each change of medium; (2) was reciprocally related to the cAMP produced; and (3) was competed for by ATPases present in added serum or by hexokinase and, less effectively, by exoenzymes on the cell surface. The extent of ATP leakage varied widely between different cell lines, being marked in CHO and Y-1 adrenal cells but negligible in transformed lymphocyte lines. The uncertainty of the origin of cAMP found in media of cultured cells requires separate analysis of cell and medium cAMP and an assessment of ATP leakage.