Myosin-I is thought to supply the force for movement of cell membranes relative to actin filaments (reviewed in refs 1, 2), but confirmation of this hypothesis has been difficult because of the presence of multiple isoforms of myosin-I and other unconventional myosins in most cells. We report here the first evidence that a myosin-I isoform is essential for a specific class of intracellular membrane movements in vivo. In Acanthamoeba, the contractile vacuole is an autonomous structure which fuses with the plasma membrane to control the water content of the cell. Because myosin-IC is the only myosin-I isoform concentrated in the contractile vacuole complex, and a protein antigenically related to myosin-IC is located on or near the Dictyostelium (slime mould) contractile vacuole, we thought this organelle might provide the best opportunity to demonstrate a relationship between myosin-I and membrane motility. Antibodies that inhibit the activity of Acanthamoeba myosin-IC in vitro interfere with expulsion of excess water by the contractile vacuole in vivo, leading to overfilling of this organelle and cell lysis. Myosin-IC may generate the force required to contract the vacuole and may also be involved in transfer of water to the contractile vacuole during refilling.