Cow milk fat triglyeerides were fractionated by low-temperature crystallization from pentane, and the —60-degree filtrate was separated further by countercurrent distribution (CCD). Representative fractions were analyzed for fatty acid composition by gas chromatography. Long-chain triglycerides migrated early in CCD and contained comparatively large amounts of C18:1 acid, which was associated predominantly with C16:0 and C18:0 acids in the glycerides. Shortchain triglycerides migrated later in CCD, and as their short-chain acid content increased there was a concurrent decrease in C18:1.Fractions representative of the short- and long-chain triglyceride groups were studied by lipase hydrolysis, and the proportion of the total amount of each fatty acid esterified at the 2-position was calculated. The results confirmed the nonrandom distribution of most of the major fatty acids in milk fat. Improved methods of separation and analysis are needed to elucidate the structure of the individual triglycerides present.