The trans fatty acid (TFA) patterns in the fats of ruminant meat and dairy products differ from those found in other (processed) fats. We have evaluated different TFA isomers in human breast milk as an indicator of dietary intake of ruminant and dairy fats of different origins. Breast milk samples were collected 1 month postpartum from 310 mothers participating in the KOALA Birth Cohort Study (The Netherlands). The study participants had different lifestyles and consumed different amounts of dairy products. Fatty acid methyl esters were determined by GC-FID and the data were evaluated by principal component analysis (PCA), ANOVA/Post Hoc test and linear regression analysis. The two major principal components were (1) 18:1 trans-isomers and (2) markers of dairy fat including 15:0, 17:0, 11(trans)18:1 and 9(cis),11(trans)18:2 (CLA). Despite similar total TFA values, the 9(trans)18:1/11(trans)18:1-ratio and the 10(trans)18:1/11(trans)18:1-ratio were significantly lower in milk from mothers with high dairy fat intake (40-76 g/day: 0.91 +/- 0.48, P < 0.05) compared to low dairy fat intake (0-10 g/day: 1.59 +/- 0.48), and lower with strict organic meat and dairy use (>90% organic: 0.92 +/- 0.46, P < 0.05) compared to conventional origin of meat and dairy (1.40 +/- 0.61). Similar results were obtained for the 10(trans)18:1/11(trans)18:1-ratio. We conclude that both ratios are indicators of different intake of TFA from ruminant and dairy origin relative to other (including industrial) sources.