Addition of varying concentrations of oxalate to isolated chicken hepatocytes reduced gluconeogenesis from lactate in a manner indicating that pyruvate carboxylase was not the rate-limiting step. With hepatocytes from biotin-deficient chicks, sensitivity to inhibition was increased, and was consistent with pyruvate carboxylase being rate-limiting. Administration of biotin to deficient chicks overnight restores sensitivity to oxalate to normal.