Previous experimentation involving the use of dispersed rat liver cells have utilized suspending media common to fractionation and slicing methods. Cells in these media have not remained viable for prolonged periods of time and they have resisted culturing techniques. Suspensions of dispersed parenchymal cells were prepared from rat livers which had been perfused in situ via the dorsal aorta with an EDTA-sucrose solution. The maintenance of surviving cells was attempted in three different media: sucrose buffered with Tris-HCl, Waymouth medium, and Waymouth medium supplemented with 30% calf serum. Cells suspended in sucrose and buffered with Tris-HCl oxidized citrate, succinate, and α-kegoglutarate but did not respire in the presence of other citric acid cycle intermediates. When cells were suspended in Waymouth medium without glucose, they oxidized malate and glutamate plus the above-mentioned substrates. Glucose and pyruvate did not stimulate oxygen uptake in either medium. Cells exhibited respiratory activity for up to 8 hr when incubated in Waymouth medium supplemented with calf serum. Both the ability to oxidize succinate and the morphological integrity of the cells were retained for this period of time. When cells were incubated in Waymouth medium alone, the time interval was reduced to 6 hr. Sucrose-Tris-HCl in the presence of succinate was not satisfactory as an incubation medium, since many of the cells underwent breakdown.