Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco 'Bright Yellow-2' cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K(+) currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E(K) when external [K(+)] was either 6 or 60 mM. Several channel blockers, including external Ba(2+), niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K(+) current. Among the monovalent cations tested (NH(4)(+), Rb(+), Li(+), Na(+)), only Rb(+) had appreciable permeation (P(Rb)/P(K) (=) 0.7). In addition, in 60 mM K(+) solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K(+) current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E(K), and other monovalent cations (NH(4)(+), Rb(+), Li(+), Na(+)) were not permeant. The inward current was blocked by external Ba(2+) and niflumic acid. External Cs(+) reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K(+) current was generally small compared to the amplitude of the outward K(+) current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K(+) currents in tobacco 'Bright Yellow-2' cells.