The time course of the glutaminase reaction in sonicated liver mitochondria exhibited a variable lag period before the final steady rate of glutamate formation was obtained. Added NH4Cl reduced the lag period without altering the final rate. Measurements of time courses under various conditions of pH and protein concentration showed that a half-maximum rate occurred at a critical concentration of NH3 which was independent of pH. It was concluded that in the pH range 7.0-8.0 liver glutaminase is activated by NH3 rather than by NH4+.