The peroxisome proliferator-activated receptors (PPAR) can play pivotal nutrigenomics roles in ruminants owing their capacity to be activated by long-chain fatty acids (LCFA). Data on expression of putative PPARs target genes suggested that palmitate (PA) is a potent PPARs activator in cows and goats whereas trans-10,cis-12 conjugated linoleic acid (CLA) is not or is a very weak PPAR agonist. Furthermore, previous in vivo data in goats and cows suggested that 2,4-thiazolidinedione (TZD) is a weak activator of PPARγ. To test if palmitate, trans-10,cis-12-CLA, and TZD are PPAR agonists we have transfected immortalized MacT and primary mammary goat (PMG) cells with a PPRE-X3-TK-Luc plasmid. Prior transfection cells were seeded at 10⁴ cells/well in a 96-well plate. We have used as transfection reagents Lipofectamine 3000 with PMG and TransIT-X2 with MacT at 0.3 μL/well with 200 ng/well of plasmid in OptiMEM deprived of fetal bovine serum (FBS). Transfected cells were treated for 24h in quadruplicates with 10% FBS or 0.1 mM of ethanol (control), rosiglitazone (ROSI), PA, and CLA as well as 2 doses of TZD (0.1 mM and 1 mM) in medium without FBS. All were supplemented with 10 μM of 9-cis-retinoic acid. Cell number and transfection efficiency was obtained via CellProfiler software and luciferase was measured using luminometer and normalized by number of viable cells. Data were analyzed using GLIMMIX of SAS and significance was declared with P < 0.05. Transfection was higher with MacT (24 ± 3%) compared with PMG (6 ± 4%). In MacT cells, all treatments with exception of CLA activated PPAR compared with control with the highest activation observed with TZD followed by PA, ROSI, and FBS. In PMG the PPAR activation was observed with FBS, PA, and TZD, while CLA and ROSI did not affect luciferase. Overall our data confirmed that TZD is a PPARγ synthetic agonist in ruminant mammary cells. In addition, the data confirmed that PA but not CLA is a PPAR activator and that blood serum in ruminants contains potent natural PPAR agonists, likely LCFA. These findings underscore the nutrigenomics importance of PA and confirmed the PPARγ responsiveness of mammary cells in ruminants.