Effects of basolateral monovalent cation replacements (Na⁺ by Li⁺, K⁺, Cs⁺, methylammonium, and guanidinium) on permeability to ⁸⁶Rb of volume-sensitive cation channels (VSCC) in the basolateral membrane and on regulatory volume decrease (RVD), elicited by a hyposmotic shock, were studied in A6 epithelia in the absence of apical Na⁺ uptake. A complete and quick RVD occurred only when the cells were perfused with Na⁺ or Li⁺ saline. With both cations, hypotonicity increased basolateral ⁸⁶Rb release (R^bl_Rb), which reached a maximum after 15 min and declined back to control level. When the major cation was K⁺, Cs⁺, methylammonium, or guanidinium, the RVD was abolished. Methylammonium induced a biphasic time course of cell thickness (T_c), with an initial decline of T_c followed by a gradual increase. With K⁺, Cs⁺, or guanidinium, T_c increased monotonously after the rapid initial rise evoked by the hypotonic challenge. In the presence of K⁺, Cs⁺, or methylammonium, R^bl_Rb remained high during most of the hypotonic period, whereas with guanidinium blockage of R^bl_Rb was initiated after 6 min of hypotonicity, suggesting an intracellular location of the site of action. With all cations, 0.5 mM basolateral Gd³⁺ completely blocked RVD and fully abolished the R^bl_Rb increase induced by the hypotonic shock. The lanthanide also blocked the additional volume increase induced by Cs⁺, K⁺, guanidinium, or methylammonium. When pH was lowered from 7.4 to 6.0, RVD and R^bl_Rb were markedly inhibited. This study demonstrates that the VSCCs in the basolateral membrane of A6 cells are permeable to K⁺, Rb⁺, Cs⁺, methylammonium, and guanidinium, whereas a marked inhibitory effect is exerted by Gd³⁺, protons, and possibly intracellular guanidinium.