Mammalian cells regulate their volume to prevent unintentional changes in intracellular signaling, cell metabolism, and DNA integrity. Intentional cell volume changes occur as cells undergo proliferation, apoptosis, or cell migration. To regulate cell volume, cells use channels and transport systems to flux osmolytes across the plasma membrane followed by the obligatory movement of water. While essentially all cells are capable of regulatory volume decrease (RVD), regulatory volume increase (RVI) mechanisms have only been reported in some cell types. In this investigation, we used human glioma cells as a model system to determine conditions necessary for RVI. When exposed to hyperosmotic conditions through the addition of 30 mosM NaCl or sucrose, D54-MG and U251 glioma cell lines and glioma cells from acute patient biopsies shrunk transiently but were able to fully recover their original cell volume within 40-70 min. This ability was highly temperature sensitive and absolutely required the presence of low millimolar concentrations of l-glutamine in the extracellular solution. Other known substrates of glutamine transporters such as methyl-amino isobutyric acid (MeAIB), alanine, and threonine were unable to support RVI. The ability of cells to undergo RVI also required the presence of Na+, K+, and Cl- and was inhibited by the NKCC inhibitor, bumetanide, consistent with the involvement of a Na+/K+/2Cl- cotransporter (NKCC). Moreover, the expression of NKCC1 was demonstrated by Western blot. We concluded that regulatory volume increase in human glioma cells occurs through the uptake of Na+, K+, and Cl- by NKCC1 and is modulated by the presence of glutamine.