The effects of various unsaturated fatty acids such as oleic (18:1n-9), linoleic (18:2n-6) and arachidonic (20:4n-6) on the activities of fatty acid synthetase (FAS), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) all were determined in primary cultures of mouse hepatocytes. Activities of FAS and ME were found to decrease with time in culture regardless of whether hepatocyte donors were fed diets containing polyunsaturated fatty acid-free hydrogenated cottonseed oil (HCTO) or corn oil (CO). On the other hand, while G6PDH activity also declined in cultured hepatocytes obtained from HCTO-fed mice, the activity of this enzyme increased in cells cultured from CO-fed mice. 6PGDH activity was found to increase in hepatocytes obtained from both diet groups. Neither 18:2 nor 20:4 when added to media could alter FAS or ME activities compared with those observed with either 18:1-containing or fatty acid-free media. Since lactic dehydrogenase activity and the rate of incorporation of [3H] leucine into FAS protein were unaltered with time in hepatocyte cultures, the decreased activities of FAS and ME cannot be attributed to a loss in cell viability during culture but rather appear to be specific for those enzymes which respond to diet hormones in vivo.