To better characterize loop diuretic-sensitive ion fluxes in the inner medullary collecting duct (IMCD) we examined them in IMCD cells grown as a primary culture on permeable supports. A polarization of the cells with their basolateral side to the support was confirmed morphologically by electron microscopy and functionally by flux studies with ouabain. Within 7 days cells developed a transepithelial resistance of 974+/-52 omega per cm2 and a low transepithelial potential difference (-0.7+/-0.8 mV). Measurements of intracellular ion content by electron probe microanalysis in IMCD depleted of intracellular ions by preincubation in a Na(+)-K(+)-Cl(-)-free medium revealed, compared to the control receiving solvent, significant reductions in intracellular Na+ content (-17.6% within 10 min) and intracellular Cl- content (-43.8% within 30 min) by the addition of bumetanide (10(-4) mol/l) to the apical but not basolateral incubation medium. In 22Na+ and 86Rb+ isotope uptake studies, fluxes from the apical side were significantly inhibited at bumetanide concentrations of 100 micromol/l by 0.27+/-0.10 and 0.21+/-0.04 nmol/cm2 in 10 min, respectively, whereas basolateral fluxes of 86Rb+ but not 22Na+ were significantly reduced by this substance. Removal of Cl- had a similar but not additional effect. mRNA encoding the apical isoform of the Na+2Cl(-)K+ cotransporter could be specifically amplified by reverse transcriptase polymerase chain reaction from the inner medulla and highly purified IMCD cells. Northern blot of mRNA isolated from the inner medulla with a riboprobe of the apical isoform revealed a transcript of approximately 4.9 kb. This probe localized under "low-stringency" conditions to the IMCD in in situ hybridization studies. These results suggest the presence of an apically localized isoform of bumetanide-sensitive Na+2Cl(-)K+ cotransport in at least a subfraction of IMCD cells. This transport may be involved in the ultimate adjustment of urinary electrolyte concentration by this final segment of the tubular system.