Incubation (30 degrees) of fat cell particulate fractions with fluoride before assay in the effective absence of fluoride results in activation of adenylate cyclase. Whereas the effect of fluoride (1.3 to 7 mM) when added to the assay was maximal in less than 2 min, 10 or 15 min of incubation before assay was usually required to produce maximal activation with any given concentration of fluoride. Under both conditions 3 to 5 mM fluoride produced maximal activation. After incubation with fluoride for 5 to 20 min cyclase activity was constant for at least 15 min of assay without fluoride; maximal activity was greater than that produced by fluoride added to the assay system and the concentration required to produce significant activation was lower. Fluoride activation in the assay or during prior incubation could be prevented by pyrophosphate. When added during the early minutes of assays with fluoride, 1.5 mM pyrophosphate, which had little effect on the activity of enzyme previously incubated with fluoride, rapidly reduced activity to essentially basal levels; when added after 10 min its effect was equally rapid but much smaller. It appears that activation is initially reversible by removal of fluoride as it is by addition of pyrophosphate, but becomes largely irreversible by these means with continued exposure to fluoride. Fluoride in the assay system inhibited cyclase activated by isoproterenol or choleragen or by incubation with fluoride prior to assay; inhibition, dependent on fluoride concentration, was maximal at 5.3 mM. Since maximal activity produced by incubation with fluoride before assay exceeded that of nonincubated preparations assayed with fluoride, and was reduced to the latter level when assayed in the presence of fluoride, we infer that inhibition is reversible at a time when fluoride activation is relatively irreversible. Pyrophosphate (1.5 mM), which prevented fluoride activation, did not reduce fluoride inhibition of isoproterenol-, fluoride-, or choleragen-activated cyclase. When 3 mM MnCl2 was present in the assay, inhibition by fluoride was not observed. In descriptive terms, MnCl2 appeared to cause rapid reversal of fluoride inhibition. Thus, fluoride inhibits, in an apparently similar manner, fat cell adenylate cyclase whether it is activated by isoproterenol, fluoride, or choleragen. Although fluoride activation and inhibition can apparently be dissociated or modified differentially, until the mechanism(s) of action of fluoride is elucidated it cannot be concluded that these are totally independent processes.