We isolated two cell clones from the wild-type Madin-Darby canine kidney cell line (MDCK) that resembles renal collecting duct epithelium. Morphology and karyotypes of the two cell clones were evaluated. The MDCK-C7 cell clone morphologically resembles principal cells (polygonal cell shape, flat), while the MDCK-C11 clone resembles intercalated cells (cuboidal cell shape, high). The diploid chromosome number of MDCK-C7 cells is 83.1 +/- 0.2 (n = 139); that for MDCK-C11 cells is 78.8 +/- 0.1 (n = 128). Culture of MDCK-C7 cells in alkaline medium (pH 7.7) induced irreversible phenotypical and genotypical alterations. Transformed MDCK-C7F cells are characterized by two abnormal (biarmed) chromosomes. In contrast, MDCK-C11 cells are not phenotypically altered by alkaline stress. In order to elucidate the role of intracellular pH (pHi) in the transformation process, we measured pHi under control conditions (pH 7.4), after 5 min exposure to alkaline stress ("acute experiment," pH 7.7) and after incubation of the cells in alkaline medium for two weeks ("chronic experiment," pH 7.7). Under control conditions, MDCK-C7 cells maintained pHi at 7.14 +/- 0.01 (n = 154) and MDCK-C11 cells at 7.01 +/- 0.01 (n = 147). Acute alkaline stress increased pHi of both cell types to similar steady-state values. Under chronic alkaline stress, MDCK-C7 cells were unable to maintain intracellular pH within normal limits exhibiting sustained alkalinization, whereas MDCK-C11 cells could successfully regulate pHi. We conclude that wild-type MDCK cells consist of two genetically distinct subpopulations with different morphology and function. Only the MDCK-C7 clone that resembles the principle cell type of renal collecting duct can be transformed by alkaline stress while the MDCK-C11 clone resists this treatment, due to efficient pHi control mechanisms.