This investigation addresses whether enriching cellular phospholipids with n-3 or n-6 fatty acids affects the process of regulatory volume decrease (RVD) in murine Ehrlich ascites tumor cells. Two weeks of dietary n-3-rich fish oil (7.5%, wt/wt) increased the ratio of eicosapentaenoic acid to arachidonic acid in cellular phospholipids compared with an olive oil control diet. Cells grown in mice fed on fish oil had an accelerated RVD response after hypotonic exposure, indicating that the volume-induced K conductance was increased. The fish oil diet furthermore resulted in an increased Cl conductance during RVD, demonstrated as an increased initial rate of cell shrinkage after addition of K ionophore to the swollen cells. The initial rate of volume recovery correlated positively with the sum of eicosanoid precursors (arachidonic acid plus eicosapentaenoic acid) (P = 0.007). Diet supplemented with n-6 fatty acids resulted in an enhanced RVD response as well. RVD was inhibited by anti-calmodulin drugs, and exogenous leukotriene D and leukotriene D were equipotent in attenuating this inhibition. We conclude that dietary polyunsaturated fatty acids result in a more effective RVD response because of an increase in the volume-induced Cl and K conductances. We propose that this is caused by an enhanced volume-induced leukotriene synthesis due to an increase in eicosanoid precursor availability.