In the terminal stages of exocytosis from permeabilised mast cells, ATP has a number of modulatory actions, although its presence (and by implication, phosphorylation) is not obligatory for secretion to occur. These effects include (1) the enhancement of the sensitivity to both of the essential effectors (Ca 2+ and guanine nucleotide); (2) the maintenance of the responsiveness of permeabilised cells; (3) restoration of responsiveness to cells rendered refractory by previous permeabilisation, and (4) induction of delays in the onset of exocytosis from permeabilised cells. We define the modulatory reactions induced by ATP by characterising their specificity to other potential phosphorylating nucleotides and their requirement for Mg 2+. GTP and AppNHp are without effect in any of the modulatory actions. ATP, ATP-γ-S, ITP, XTP, CTP and UTP all appear to support an enhancement of the sensitivity to GTP-γ-S when applied immediately at the time of permeabilisation. However, the non-adenine nucleoside triphosphates appear to mediate their effect by transphosphorylation to ADP, and therefore the active species appears to be ATP. Only ATP is capable of maintaining and restoring responsiveness (2 and 3 above). Only ATP and ATP-γ-S induce onset delays and do so moreover in the absence (< 10 −8 M) of Mg 2+. We conclude that three of the modulatory effects (1, 2 and 3 above) which all express a requirement for Mg 2+, and can be prevented by inhibitors of protein kinase C are likely to result from phosphorylation reactions. The induction of delays by ATP is unlikely to incur phosphorylation.