Production of dairy products with increased amounts of nutraceutic FA such as conjugated linoleic acid (CLA) represents a recent approach for dairy producers and processors to increase the value of their products. The effect of CLA and other FA on the expression of diacylglycerol acyltransferase-1 (DGAT-1) and DGAT-2, and DGAT activity were investigated in bovine mammary gland epithelial (MAC-T) cells. DGAT gene expression analyses were also conducted using bovine mammary gland tissue from dairy cows. In the studies with MAC-T cells, there were no significant effects of CLA isomers or other FA on DGAT1 expression, whereas all FA tested showed enhanced DGAT2 expression (P < 0.05 to P < 0.001), with alpha-linolenic acid (alpha-18:3) having the greatest effect. Additionally, DGAT2 expression was co-ordinated with expression of lysophosphatidic acid acyltransferase (LPAAT), an observation that was also apparent in mammary gland from lactating dairy cows. In contrast, treatment of MAC-T cells with trans-10, cis-12 18:2 or alpha-18:3 resulted in a significant (P < 0.05) decrease in overall DGAT enzyme activity, although the mechanisms resulting in these effects are unclear. Competition assays using microsomes from bovine mammary gland tissue and 1-[(14)C]oleoyl-CoA suggested that DGAT activity was more selective for oleoyl (cis-9 18:1)-CoA than cis-9, trans-11 18:2-, trans-10, cis-12 18:2- or cis-9, cis-12 18:2-CoA. Collectively, the results suggest the relationship between trans-10, cis-12 18:2 and reduced TAG production in bovine milk is not linked to the production of DGAT1 or DGAT2 transcripts, but probably involves effects of this CLA isomer at events beyond transcription, such as post-translational and/or enzyme activity effects.