Role of cytoplasmic concentration of free Ca2+ ([Ca]in) and arachidonic acid in potentiating the effect of vitamin E (DL-alpha-tocopherol) on acetylcholine receptor activity in Helix pomatia neurons was studied using a two-microelectrode intracellular recording, voltage clamp and fluorescent calcium probe fura-2 technique. Elevation of [Ca]in by intracellular injection from a microelectrode or by depolarizing pulses and application of 0.1 microM-0.1 mM vitamin E enhanced the acetylcholine-induced chloride current both in LP11 and RBc4 neurons. Application of 10 microM arachidonic acid to the same neurons decreased this current. The use of fluorescent probe showed that vitamin E did not essentially change [Ca]in, but an increase of [Ca]in intensified both the enhancing effect of vitamin E and the depressing effect of arachidonic acid. The enhancing effect of calcium influx was considerably decreased after vitamin E application. The antioxidant action of vitamin E was probably not involved in the mechanism of its enhancing effect on acetylcholine-induced current, since synthetic antioxidant, ionol, depressed acetylcholine responses. A spectrum analysis has shown the interaction between vitamin E and arachidonic acid in solution. This interaction may be considered as the molecular mechanism responsible for the prevention by vitamin E of steady arachidonic acid production from membrane phospholipids and its down-regulatory effect on acetylcholine receptor activity. Our results support this suggestion, since an inhibitor of phospholipase A2, 4-bromophenacyl bromide, mimicked the enhancing effect of vitamin E.