The ability of isolated pea (Pisum sativum cv. Ran 1) mesophyll protoplasts to aggregate and fuse spontaneously was examined. Aiming to define whether Ca2+released during enzymatic isolation of protoplasts and residual in the purified suspension is important for aggregation and fusion, different isolating media were used: Ca2+—free medium, buffered Ca2+—free medium, Ca2 +—free medium containing 2.5 mM EGTA, and medium containing 10 mM CaCl2. Light scattering was measured to assess aggregation and/or fusion and Arsenazo III was used to evaluate free Ca2+. Binding of Ca2+to plasmalemma was characterized by 9-Aminoac ridine fluorescence. Since light scattering of purified suspensions of protoplasts isolated in the presence of 2.5 mM EGTA was lower as compared to the other variants, a Ca2+—dependent fusion during isolation was suggested. Binding of Ca2+by 100 μM EGTA slightly reduced the light scattering of suspension of protoplasts isolated without EGTA, indicating existence of Ca2+dependent aggregation. Titration with CaCl2(up to 100 mM) completely reduced protoplasts surface potential but failed to increase the aggregation significantly. The results suggested that protoplasts fuse spontaneously in the presence of Ca2+released by the tissue during isolation. In purified suspensions residual micromolar amount of Ca2+is sufficient for aggregation.