Rainbow trout (Oncorhynchus mykiss) hepatocytes were cultured under simulated conditions of varying nutritional status to explore the short-term modulation by dietary substrates of the main lipogenic enzymes: glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACoAC) and fatty acid synthetase (FAS). Primary cultures were individually exposed to varying amounts of glucose, hydrolysed casein and long-chain polyunsaturated fatty acids (PUFA) for 12 h. A second set of experiments was designed to evaluate the effects of mixing different relative amounts of these macronutrients in the culture medium. Glucose concentrations of up to 20–25 mM SHOWED A STIMULATORY EFFECT ON G6PD, ME, ACL AND ACOAC ACTIVITY (P<0·05) WHILE AN EARLIER INHIBITORY EFFECT ON FAS WAS OBSERVED AT 10–20 Mm glucose (P<0·05). The use of hydrolysed casein as a nutritional source of amino acids inhibited the activity of FAS and ME (P<0·05), and stimulated G6PD, ACoAC and ACL activity (P<0·05). Low levels of linolenic acid exerted a stimulatory effect on all the lipogenic enzymes assayed (P<0·05) with the exception of FAS, and increased amounts showed some inhibition of lipogenic activities (P<0·05). Eicosapentaenoic acid and docosahexaenoic acid showed a similar effect, although the former strongly inhibited FAS activity while the latter showed greater potential to inhibit ACoAC and G6PD. A complete change in the relative levels of glucose, hydrolysed casein and PUFA in turn led to changes in the enzyme activity patterns observed. The present study shows the feasibility of exploring the direct regulation of lipogenesis in isolated fish cells by varying the relative amounts of main macronutrients, mimicking in vivo dietary conditions. It is felt that such an approach may serve to investigate the macronutrient regulation of other metabolic pathways.