Cultured medullary thick ascending limb (MTAL) cells may lack some of the main carriers of fresh MTAL cells, such as apical Na+-K+(NH4+)-2Cl- cotransporter (BSC-1) and Na+/H+ exchanger (NHE-3). We have developed a technique to maintain rat MTALs several hours in suspension and in a good state of viability. Medullary thick ascending limbs were suspended in a 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's essential medium (HDMEM) supplemented with 25 mM HCO3- and gassed with 95% O2/5% CO2; the resulting mixture was placed in a rotary shaking water bath at 37 degrees C for 16 hours. As seen by electron microscopy, MTALs from the HDMEM-suspension retained a virtually normal tubular organization. Na+-K+(NH4+)-2Cl- cotransport activity and NHE consistent with both apical NHE-3 and basolateral NHE-1 activities were underscored both in intact cells by intracellular pH measurements and in a membrane fraction enriched in apical and basolateral membranes by 22Na+ uptake experiments. These results demonstrate that freshly harvested MTALs can be maintained in a well differentiated state for at least 16 hours; this preparation should make long-term in vitro studies of MTAL transport regulations possible.