Culturing lactating mammary explants allows evaluating the effect of bioactive molecules in a controlled environment while maintaining the same characteristics found in vivo (Keys et al., 1997; In Vitro Cell. Dev. Biol. Animal, 33:206–21). However, studies using ovine mammary tissue are still scarce. The objective of this study was to evaluate the effect of trans-10,cis-12 CLA on the expression of key genes involved in milk fat synthesis in mammary explants cultured in vitro. Mammary samples were obtained through biopsies in lactating ewes (120 DIM) and were grown on plates with growth area of 1.9 cm² in mammary epithelial cell growth media supplemented with fetal bovine serum, antibiotics, insulin and growth factors at 37°C with 5% CO₂ and humidity saturated. Tissues were cultured for 3 and 24h in triplicates using the following treatments: 75 µmol/L of trans-10,cis-12 CLA or Control (no CLA). After cultures were stopped RNA was extracted, cDNA synthesized and qRTPCR performed. The genes studied were SREBP1 (sterol regulatory element-binding protein), PPARγ (peroxisome proliferator-activated receptor gamma) and SCD1 (stearoyl-CoA desaturase). Statistical analysis was performed using the MIXED procedure of SAS (2002) and ribosomal protein S18 housekeeping gene was used as a covariate in the model. CLA treatment had no effect on gene expression of SREBP1, PPARγ, and SCD1 at 3 h (P = 0.19, P = 0.52 and P = 0.20, respectively). At 24 h, there was a trend for CLA-treated explants to have reduced gene expression of SREBP1 and PPARγ (P = 0.07 and P = 0.08, respectively), and a 12-fold decrease in SCD1 gene expression (P = 0.009). Overall, our results suggest that trans-10,cis-12 CLA downregulates expression lipogenic genes in ovine mammary tissue, however explants may need to be cultured for at least 24 h