AbstractThe mutation rate from asparagine requirement to asparagine non‐requirement has been measured using the Luria‐Delbrück Fluctuation test in the Walker 256 rat carcinosarcoma line grown in vitro. Non‐requiring variants are pre‐existing in the population, and are not induced by the selecting agent. The mutation rate was of the order of 1.4 × 10−6 per cell per generation to 3.5 × 10−6 per cell per generation. The relationship between the malignancy of the cell line and its unusual requirement for asparagine is discussed.