Palytoxin (PTX), isolated from the marine soft coral Palythoa tuberculosa, increases the cation conductance of human red cell membranes. In the presence of 10(-10) M PTX and 10(-5) M DIDS, the membrane potential approximates the equilibrium potential for Na+ or K+ rather than Cl-. Even in the absence of DIDS, the Na+ and K+ conductances were greater than the Cl- conductance. The selectivity of the PTX-induced cation conductance is K+ > Rb+ > Cs+ > Na+ > Li+ much greater than choline+ > TEA+ much greater than Mg2+. Measurements of K+ efflux revealed two apparent sites for activation by PTX, one with a K(a1), of 0.05 nM and a maximum flux, v(max1), of 1.4 mol/liter of cells per h and another with a K(a2) of 98 nM and a v(max2) of 24 mol/liter of cells per h. These effects of PTX are completely blocked by external ouabain (300-mu-M) and prevented by internal vanadate (100-mu-M). When the PTX channels are open, the Na,K pumps do not catalyze ATP hydrolysis. Upon thorough washout of cells exposed to about five molecules of PTX/pump, the Na,K pump of these cells operates normally. Blockage of the positively charged NH2 terminus of PTX with a p-bromobenzoyl group reduces the potency of the compound to induce Na and K fluxes by at least a factor of 100, and to compete with the binding of [H-3]ouabain by at least a factor of 10. These data are consistent with the conclusion that PTX binds reversibly to the Na,K pumps in the red cell membrane and opens a (10-pS) channel equally permeable to Na and K at or near each pump site.