Like voltage-operated Ca(2+) channels, store-operated CRAC channels become permeable to monovalent cations in the absence of external divalent cations. Using the whole-cell patch-clamp technique, we have characterized the permeation and selectivity properties of store-operated channels in the rat basophilic leukemia (RBL-1) cell line. Store depletion by dialysis with InsP(3) and 10 mM EGTA resulted in the rapid development of large inward currents in Na(+)- and Li(+)-based divalent-free solutions. Cs(+) permeated the channels poorly (P(Cs)/ P(Na)=0.01). Trimethylamine (TMA(+)), tetramethylammonium (TeMA(+)), tetraethylammonium (TEA(+)), N-methyl- D-glucamine (NMDG(+)) and TRIS(+) were not measurably permeant. NH(4)(+) was conducted well. We estimated the minimum pore diameter under divalent-free conditions to be between 0.32 nm and 0.55 nm. When cells were dialysed with buffered Ca(2+) solution and I(CRAC) activated by application of thapsigargin, P(Cs)/ P(Na) was still low (0.08). Outward currents through CRAC channels were carried by intracellular Na(+), K(+) and, to a much lesser extent, by Cs(+). Currents were unaffected by dialysis with Mg(2+)-free solution. The Na(+) current was inhibited by external Ca(2+) (half-maximal blocking concentration of 10 microM). This Ca(2+)-dependent block could be alleviated by hyperpolarization. The monovalent Na(+) current was voltage dependent, increasing as the holding potential depolarized above 0 mV. Our results suggest that CRAC channels in RBL-1 cells have a smaller pore diameter than voltage-operated Ca(2+) channels, discriminate between Group I cations, and differ markedly in their selectivity from CRAC channels reported in lymphocytes.