Many of the differentiated functions of hepatocytes are lost in culture, yet addition of certain medium supplements can aid in the retention of differentiated character. Therefore, the effect of time in monolayer culture on rat hepatocyte glutathione (GSH) synthesis and sensitivity to the GSH detoxicated xenobiotic ethacrynic acid was examined in cultures with and without medium supplementation by transferrin and sodium selenite. GSH content was found to be about 12 nmol/#g DNA at 4 hr in culture and to approximately triple by 24 hr. lntracellular GSH levels continued to increase in transferrin/sodium selenite-supplemented cultures, from 32 to 41.6 nmol/ l.tg DNA, while GSH levels in unsupplemented cultures declined to 18 nmol/~g DNA. However, the rate of GSH synthesis after diethylmaleate depletion was found to decrease from 4.2 to 2.8 nmol/hr/#g DNA at 4 and 24 hr after inoculation, respectively. GSH repletion rate increased to 3.9 nmol/hr/#g DNA at 48 hr. The GSH accumulation rate after depletion in supplemented cultures did not vary significantly over the initial 48 hr. Incubation for 3 hr with 100 #M ethacrynic acid (EA) did not elicit an increase in LDH leakage in hepatocyte monolayers after 4 or 48 hr in culture or in cultures with supplemented medium at any time point tested. Cultures 24 hr in medium without transferrin/sodium selenite supplementation exhibited significant LDH leakage after 3 hr of EA treatment. Over the 3 hr EA treatment, intracellular GSH content was decreased in all cultures. Only in the 24 hr unsupplemented cultures did GSH depletion exceed the 90% level previously asso-Meredith ciated with depletion of the mitochondrial pool of GSH and EA toxicity in hepatocytes. The experiments show that during the redifferentiation of hepatocytes in culture, a transient period occurs when apparent GSH synthesis is depressed and enhanced sensitivity to GSH-detoxicated compounds is observed. This period of increased sensitivity is prevented or at least delayed by inclusion of supplemental transferrin and sodium selenite, suggesting that redifferentiation can be regulated by extracellular influences.