1. Multiple conductance level ion channels were recorded in excised and cell-attached patches from cells of a mouse B lymphocyte hybridoma line. The reversal potential for the single-channel current was unaffected by the species of cation on the cytoplasmic face of the patch, but changed as the Cl- concentration was altered, indicating that the channel is anion selective. 2. The permeability sequence determined from reversal potentials was F- greater than I- greater than SCN- greater than Br- greater than Cl- greater than glucuronate greater than NO3- greater than aspartate. This was different from the conductance sequence (Cl- greater than SCN- = F- greater than Br- greater than NO3- greater than I- greater than glucuronate greater than aspartate), indicating interaction of ions within the pore of the channel. Consistent with this was the observation of anomalous mole fraction dependence with a mixed solution of thiocyanate and chloride. 3. In addition to the main open level (about 400 pS; excised patch, symmetrical 165 mM-Cl-), three subconductance levels and one supraconductance level were observed. These were concluded to be integral components of the same channel based on coincidence of appearance and identical permeabilities. 4. The channel is voltage dependent, with open probability in excised patches increasing with more positive potentials. The channel was reversibly blocked in a voltage-dependent manner by SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid), a stilbene derivative, on the cytoplasmic face. 5. Several differences were noted between cell-attached and excised-patch recordings. The multiple conductance level channel was less frequently seen in cell-attached patches but could often be induced to appear by prolonged application of positive voltages. This induced channel in attached patches showed an altered voltage dependence which could be partially mimicked in excised patches by including cyclic AMP and ATP in the solution on the cytoplasmic side of the membrane.