Abstract Electrofusion of evacuolated and vacuolated oat leaf protoplasts is difficult because of the different size and density of these cells which results in separation of the two fusion partners during dielectrophoresis. The fusion yield of this cell system was considerably enhanced by electrofusion in hypo-osmolar media containing 0.4 M mannitol, 0.1 mM calcium acetate and 0.1% bovine serum albumin. This increase in yield was only achieved if the dielectrophoretically induced membrane contact between the two fusion partners was enhanced by an initial short ‘burst’ of higher field strength (500 V/cm, peak to peak, for 5 s followed by a reduction of to 90 V/cm, peak to peak, for 20 s, frequency 1 MHz). Due to the high field strength of the alternating field at the beginning of cell chain formation separation of fusion partners of different size and density was mainly avoided. Simultaneously, the short duration of this high field ‘burst’ avoided the generation of lethal effects in the cell membranes. The subsequent low field strength of the alternating field was sufficient to keep the aligned cells in position. Optimum fusion was induced by a single square pulse of 750 V/cm and 30 μsec duration. The time required for rounding up of the heterologous fusion products decreased with decreasing osmolarity. Fusion resulted in a 5.7 ± 1.2% yield of heterologous fusion products (compared to 0.7% using the conventional electrofusion protocol) as determined by flow cytometric assay. About 50% of the vacuolated oat protoplasts and 20–50% of the heterologous fusion products regenerated their cell walls within 5 days after hypo-osmolar treatment, but no cell divisions could be observed. Evacuolated oat protoplasts died after 2–3 days in culture without any detectable cell wall regeneration.