Fusion of phagolysosomes has been previously demonstrated to occur during the incubation of phagolysosome-containing homogenates of Acanthamoeba (Oates and Touster, 1978, J. Cell Biol. 79:217-234). Further studies on this system have shown that methylxanthines (0.2 mM) and/or cAMP (0.5-1 mM) markedly accelerate the average rate, but not the extent, of the in vitro phagolysosome fusion process. Adenosine, 5'-AMP, and ADP (0.5-1 mM) were without effect. ATP (0.5-1 mM) caused variable stimulation, whereas beta, gamma-methylene-ATP (1 mM) caused pronounced inhibition, as did GTP (1 mM) and cGMP (1 mM). Stimulation by 3-isobutyl-1-methylxanthine was blocked by GTP, but not by ATP or cAMP. These results indicate that the rate of phagolysosome fusion in Acanthamoeba homogenates may be regulated by cyclic nucleotides, with enhancement of the fusion rate by cAMP and inhibition of the rate by cGMP. The extent of the reaction increased spontaneously and markedly during the first few hours after preparation of the homogenates. This activation appears to be because of a slow conversion of a significant fraction of the vacuole population from a fusion-incompetent to a fusion-competent, cyclic nucleotide-sensitive state.