This study was carried out to optimize the concentration of Ca2+ and pH of fusion medium which affected electrofusion frequency of protoplasts isolated from Nicotiana tabacum L. (cv. BY4) mesophyll cells and callus. The protoplasts were electrofused in the fusion media containing two different Ca2+ concentrations and three different pH regions. Fusion frequency was lower in the fusion medium containing only 13% mannitol as osmotic stabilizer. However, higher degree of fusion frequency (47.3%) was observed in the fusion medium containing 50mM CaCl2 at pH 10.5 than any other conditions. Cell viability was decreased by Ca2+ and high pH treatment in the fusion media, while fusion frequency was increased. It is concluded that Ca2+ is involved in electrofusion of protoplasts.