This study explores the potential use of stable carbon isotope ratios (δ(13)C) of single fatty acids (FA) as tracers for the transformation of FA from diet to milk, with focus on the metabolic origin of c9,t11-18:2. For this purpose, dairy cows were fed diets based exclusively on C(3) and C(4) plants. The FA in milk and feed were fractionated by silver-ion thin-layer chromatography and analyzed for their δ(13)C values. Mean δ(13)C values of FA from C(3) milk were lower compared to those from C(4) milk (-30.1‰ vs. -24.9‰, respectively). In both groups the most negative δ(13)C values of all FA analyzed were measured for c9,t11-18:2 (C(3) milk = -37.0 ± 2.7‰; C(4) milk -31.4 ± 1.4‰). Compared to the dietary precursors 18:2n-6 and 18:3n-3, no significant (13)C-depletion was measured in t11-18:1. This suggests that the δ(13)C-change in c9,t11-18:2 did not originate from the microbial biohydrogenation in the rumen, but most probably from endogenous desaturation of t11-18:1. It appears that the natural δ(13)C differences in some dietary FA are at least partly preserved in milk FA. Therefore, carbon isotope analyses of individual FA could be useful for studying metabolic transformation processes in ruminants.