Effects of the osmotic pressure of culture medium on the membrane fusion of L929 cells in the monolayer state were investigated using poly(ethylene glycol) (PEG) with the molecular weight of 3,000 at various concentrations at phosphate buffer saline (PBS). Cell incubation for fusion was performed via three stages; (1) incubation before PEG treatment (preincubation), (2) incubation in the presence of PEG (PEG incubation), and (3) incubation after PEG treatment (postincubation). The PBS concentrations half that of a isotonic solution in the pre- and postincubation stages significantly accelerated the membrane fusion, whereas cell treatment at more hypotonic or hypertonic concentrations of PBS suppressed cell fusion. This result was explained in terms of cell swelling and shrinking induced by the osmotic pressure difference, because such cell morphological changes actually occurred when the PBS concentration was varied from the isotonicity. In contrast, almost no effect of osmotic pressure on cell fusion was observed if PEG was present in the culture medium at 40 w/w% concentration, regardless of the PBS concentration.