RabD, a Dictyostelium Rab14-related GTPase, localizes in the endo-lysosomal pathway and contractile vacuole system of membranes. Cell lines expressing dominant-negative RabD were defective in endocytosis, endosomal membrane flow and homotypic lysosome fusion. In support of a role for RabD in fusion, cells overexpressing constitutively active RabD(Q67L) accumulated enlarged hydrolase-rich acidic vesicles ringed with GFP-RabD, consistent with RabD directly regulating lysosome fusion. To determine whether RabD also regulated phagocytosis and/or homotypic phagosome fusion (a process stimulated by many intracellular pathogens), cells overexpressing dominant-active (RabD(Q67L)) or dominant-negative (Rab(N121I)) RabD were analyzed microscopically and biochemically. The rate of phagocytosis was increased two-fold in RabD(Q67L)-expressing cells and reduced by 50% in RabD(N121I)-expressing cells compared with control cells. To examine the role of RabD in the formation of multiparticle phagosomes, we performed a series of pulse-chase experiments using fluorescently labeled bacteria and fluorescent latex beads. The rate of fusion of newly formed phagosomes was five times higher in the RabD(Q67L)-expressing cells and reduced by over 50% in RabD(N121I)-expressing cells as compared with control cells. GFP-RabD(Q67L) was found to ring multiparticle spacious phagosomes, which supports a direct role for this protein in regulating fusion. Inhibition of PI 3-kinase activity, which is known to regulate phagosome fusion in the wild-type cells, reduced the rate of phagosome fusion in RabD(Q67L+) cells, indicating that RabD acted upstream of or parallel with PI 3-kinase. We hypothesize that RabD and, possibly, Rab14, a related GTPase that associates with phagosomes in mammalian cells, are important regulators of homotypic phagosome and endo-lysosome fusion.