A quantitative trait locus (QTL) for milk fat percentage has been mappedconsistently to the centromeric region of bovine chromosome 14 (BTA14). Twoindependent studies have identified the nonconservative mutation K232A in theacylCoA-diacylglycerol-acyltransferase 1 (DGAT1) gene as likely to be causal forthe observed variation. Here we provide evidence for additional geneticvariability at the same QTL that is associated with milk fat percentagevariation within the German Holstein population. Namely, we show that alleles ofthe DGAT1 promoter derived from the variable number of tandem repeat (VNTR)polymorphism are associated with milk fat content in animals homozygous for theallele 232A at DGAT1. Our results present another example for more than twotrait-associated alleles being involved in a major gene effect on a quantitativetrait. The segregation of multiple alleles affecting milk production traits atthe QTL on BTA14 has to be considered whenever marker-assisted selectionprograms are implemented in dairy cattle. Due to the presence of a potentialtranscription factor binding site in the 18mer element of the VNTR, thevariation in the number of tandem repeats of the 18mer element might be causalfor the variability in the transcription level of the DGAT1 gene. Copyright 2004Genetics Society of America