Alanine metabolism in normal and pyridoxine-deficient rats was studied in vivo and in vitro. Incorporation of 14C-alanine into various liver components was determined and no difference was shown between normal and deficient animals in the incorporation into liver homogenates, lipid, protein and plasma glucose. Using the liver slice syste, gluconeogenic activity from alanine or pyruvate was 40% lower in deficient rats compared with the activity of normal rats. However, inhibition was completely removed by the addition of 2-oxoglutarate to alanine. Penicillamine did not affect glucose formation alanine in the liver slice.