Conditions for maintaining the activity of alcohol dehydrogenase in cultures of hepatocytes isolated from female rats were studied. The activity of alcohol dehydrogenase in freshly isolated cells was 1.7 U/mg DNA. When cultured, the activity declined 20% after one day of culture, irrespective of the culture conditions. In a conventional medium with 5 mM glucose the activity after one week of culture was only 30% of that initially measured in culture. Addition of 25 mM glucose or a high concentration of amino acids delayed the decrease. When these compounds were added together it was possible to maintain the initial activity for one week, but the activity declined during the following week. Addition of growth hormone had no effect during the first week of culture but abolished the fall during the second week. The initial metabolism of ethanol was 0.65 mumol/min x mg DNA and declined to two-thirds during the 2 weeks of culture.