The milk from individual cows was analyzed by gas chromatography (GC) of the butyl esters and their indices of atherogenicity (IA) were determined. Butter samples were made from high-IA, low-IA, and bulk-tank milk. The IA values, fatty acid profile, and triacylglycerol composition were determined by GC. As judged by the sensory panel, the IA differences of the milks and the butters had no detectable impact on the butter flavor as judged by the sensory panel. The hardness/softness was tested for butter samples in three IA classes. In term of penetration and creep, low-IA butter and high-IA butters were significantly different in texture. The cis and trans isomers of fatty acids from the butter samples were analyzed by making methyl esters from butterfat and silver-ion thin-layer chromatography (TLC). The trans-18:1[Superscript Delta]11 was the most abundant trans fatty acid in butter. The proportion of trans-18:1[Superscript Delta]11 to cis-18:1 was about the same for high-IA butter and low-IA butter. The trans- 16:1 acids are relatively higher for high-IA butter compared to cis-18:1. The triacylglcerol from butterfat was fractionated into eight bands by silver-ion TLC. The fatty acid composition of the TAG from each band was analyzed by GC. The shortest saturated fatty acid C4:0 appeared from band 2, which means C4:0 was absent in fully saturated TAG molecules. The C18:1 began to appear in band 2, then increased to become the most abundant fatty acid from band 5 to band 9. The bulk butter values tend to fall between these of high- and low-IA. During IA determination of the milks, we found some low IA milk samples that had a separated peak that eluted just before cis-18.1 in the gas chromatograph. This peak was found in the trans monoene band by silver-ion chromatography. In order to identify the position of the double bond in this fatty acid, a picolinyl ester derivative of this fatty acid mixture was made and analyzed by GC/MS. The fragmentation pattern suggests that the double band was located between carbon 11 and carbon 12 counting from the carboxyl end of the fatty acid. Thus, the mass spectrum represents vaccenic acid, trans-11-octodecenoic acid.