Carp respond to cold by the upregulated expression of 9-acyl-CoA desaturase. Here we report the cloning and characterization of Cds2, a second 9-acyl CoA-desaturase expressed in carp liver. Both Cds1 and Cds2 complemented the ole1 mutation in Saccharomyces cerevisiae, permitting the synthesis of 9-monounsaturates, confirming their identity as 9-desaturases. We demonstrate that under a standard feeding regime it is the Cds2, and not Cds1, transcript that is transiently upregulated during the first few days of cooling from 30°C to 10°C, the period when cold-induced membrane restructuring occurs. Cds2 exists as two differentially spliced transcripts, differing by a small segment from the 3'-untranslated region, the ratio of which varies with temperature. Feeding a diet enriched in saturated fats produced a fourfold increase in Cds1 transcript levels, which was blocked by cooling to 15°C. Cds2 transcript levels, however, showed no substantial response to the saturated diet. Thus carp liver uniquely expresses two isoforms of 9-acyl CoA desaturase, possibly formed by a recent duplication event, that are differentially regulated by cooling and dietary treatment.