This paper demonstrates that specific chymotryptic digestion of the cytoplasmic domain of the beta subunit of Na/K-ATPase leads to changes in the kinetics of occlusion of Rb+ ions. The experiments utilize extensively trypsinized Na/K-ATPase, "19-kDa membranes," which lack cytoplasmic loops of the alpha subunit, whereas membrane-embedded fragments (a COOH-terminal 19 kDa and three fragments of 8.1-11.7 kDa) containing transmembrane segments and extracellular loops are intact. The beta subunit is partially split into NH2- and COOH-terminal fragments of 16 and approximately 50 kDa, respectively. Cation occlusion and ouabain binding are preserved. The 19-kDa membranes were incubated, at 37 degrees C, with a selection of proteases, in the presence of Rb+ ions. In these conditions, only alpha-chymotrypsin destroyed the ability to occlude Rb+ ions. This process was associated with truncation of the 16-kDa fragment of the beta subunit in two stages. In the first stage, chymotrypsin removed 10 residues from the 16-kDa fragment to form a 15-kDa fragment (NH2-terminal Ile15) and 4 or 6 residues from the NH2 terminus of the alpha subunit fragment beginning at Asp68. In these membranes Rb+ occlusion was still intact at 37 degrees C. Strikingly, however, deocclusion of two Rb+ ions, which is characteristically biphasic in 19-kDa membranes, displayed deocclusion kinetic with mainly one fast phase. These membranes also showed a much lower affinity for Rb+ ions compared with 19-kDa membranes; and, consistent with the lower Rb+ affinity, Rb+ ions, at nonsaturating concentrations, protected less well against thermal inactivation of Rb+ occlusion. In the second stage, the 15-kDa fragment was truncated further to a 14-kDa fragment (NH2-terminal Leu24), followed by thermal destabilization of Rb+ occlusion even at high concentrations of Rb+ ions. Eventually, the thermally inactivated complex of fragments of alpha and beta subunits was digested to the limit peptides. The results suggest that the cytoplasmic domain of the beta subunit interacts with that of the alpha subunit, possibly with residues leading into the first transmembrane segment, and controls access of Rb+ ions into or out of the occlusion sites.