DGAT1 encodes diacylglycerol O-acyltransferase (EC ), a microsomal enzyme thatcatalyzes the final step of triglyceride synthesis. It became a functionalcandidate gene for lactation traits after studies indicated that mice lackingboth copies of DGAT1 are completely devoid of milk secretion, most likelybecause of deficient triglyceride synthesis in the mammary gland. Our mappingstudies placed DGAT1 close to the region of a quantitative trait locus (QTL) onbovine chromosome 14 for variation in fat content of milk. Sequencing of DGAT1from pooled DNA revealed significant frequency shifts at several variablepositions between groups of animals with high and low breeding values for milkfat content in different breeds (Holstein-Friesian, Fleckvieh, and Braunvieh).Among the variants was a nonconservative substitution of lysine by alanine(K232A), with the lysine-encoding allele being associated with higher milk fatcontent. Haplotype analysis indicated the lysine variant to be ancestral. Twoanimals that were typed heterozygous (Qq) at the QTL based on marker-assistedQTL-genotyping were heterozygous for the K232A substitution, whereas 14 animalsthat are most likely qq at the QTL were homozygous for the alanine-encodingallele. An independent association study in Fleckvieh animals confirmed thepositive effect of the lysine variant on milk fat content. We consider thenonconservative K232A substitution to be directly responsible for the QTLvariation, although our genetic studies cannot provide formal proof.