The culture for 7 days in medium with 5.5 mM glucose and 1 mM 2-deoxy-D-glucose enhanced the glucose sensitivity of neonatal rat B cells, and even stimulated their growth in vitro. Also, 2-deoxy-D-glucose supplementation maintained insulin release evoked by leucine and 2-ketoisocaproate from B cells at day 7 at levels several times higher than at day 1. The effect of leucine was greatly augmented by glutamine, whereas that of the 2-keto acid remained almost unchanged irrespective of the presence of glutamine. These results suggest an increase in oxidative catabolism of medium nutrients in B cells grown in medium with 2-deoxy-D-glucose for 7 days, and such metabolic changes may promote the growth of B cells in vitro.