The effects of loop diuretics and ion substitution on the 2-min uptake of K (86Rb as marker) were analyzed to obtain evidence for K cotransport with Na and Cl in the choroid plexus epithelium. The isolated plexuses, which were excised from lateral ventricles of adult rats, were bathed in artificial cerebrospinal fluid (aCSF). At concentrations of 10(-6) to 10(-4) M, the specific cotransport inhibitors, bumetanide and piretanide, suppressed uptake of K in a dose-dependent manner. Ouabain-insensitive K uptake was stimulated by preincubating the choroid plexus in aCSF very low in [Na] and [K], then incubating it in much higher concentrations of these cations; bumetanide (10(-4)M) blocked this stimulated uptake by 52%. Moreover, tissue preincubation in Na- or Cl-free medium, followed by incubation with normal concentrations of both ions, stimulated the ouabain-insensitive uptake of K from 15 (baseline) to 35 nmol/mg dry weight. This stimulation of K transport depended on the simultaneous presence of both Na and Cl in aCSF, and replacing either ion alone did not stimulate the ouabain-insensitive K uptake. Collectively, these findings, together with those from a previous pharmacological study of 22Na and 36Cl transport, constitute strong evidence for the cotransport of Na, K, and Cl in rat choroid plexus.