The goal of this study was to assess the effect of extracellular glucose and K⁺ ([K]_o) on the intracellular osmolyte content and cell volume maintenance and regulation in a human embryonic kidney cell line (tsA201a). Cell volume maintenance was studied by isotonic (313  ±  5 mOsm) replacement of culture media by a glucose-free Ringer solution containing (in mM) 0, 3, 6, or 10 K⁺. Cell volume regulation was studied by exposing cells to hypotonic (250  ±  5 mOsm) glucose-free Ringer solution containing the various [K]_o. The results showed that: 1) intracellular osomlyte content (i.e. Na⁺, Cl^-, Urea and free amino acids (FAA)) and cell volume increased when culture media was replaced with isotonic Ringer at all [K]_o; 2) osmolyte content decreased with continuous exposure to isotonic Ringer at all [K]_o but cell volume changes depended on [K]_o. Volume recovery occurred at 6 and 10 mM K⁺; 3) exposure to hypotonic Ringer induced swelling at all [K]_o followed by a reduction in measured intracellular osmolytes. Regulatory volume decrease occurred in 6 or 10 mM K⁺ but swelling continued in 0 or 3 mM K⁺; and 4) addition of ouabain produced swelling without recovery under iso- and hypotonic conditions. These results indicate that the removal of extracellular glucose produced a transient inhibition of the Na⁺/K⁺ ATPase resulting in a transient increase in the intracellular content of Urea, FAA and cell volume and [K]_o regulated an as yet unidentified intracellular osmolyte.