Various QTL mapping experiments led to the detection of a QTL in the centromericregion of cattle chromosome 14 that had a major effect on the fat content ofmilk. Recently, the gene encoding diacylglycerol O-acyltransferase (DGAT1) wasproposed to be a positional and functional candidate for this trait. This studyinvestigated the effects of a nonconservative lysine to alanine (K232A)substitution in DGAT1, which very likely represents the causal mutation, on milkproduction traits. Existing granddaughter designs for Fleckvieh and GermanHolstein, the two major dairy/dual-purpose breeds in Germany, were used toestimate allele frequencies and gene substitution effects for milk, fat, andprotein yield, as well as fat and protein content. A restriction fragment lengthpolymorphism assay was applied to diagnose the K232A substitution in DGAT1.Estimates of the allele frequencies for the lysine-encoding variant were basedon maternally inherited alleles in sons and amounted to 0.072 for Fleckvieh and0.548 for German Holstein. Effects of DGAT1 variants on content traits werepronounced; estimates of the gene substitution effect for the lysine-encodingvariant were 0.35 and 0.28% for fat content and 0.10 and 0.06% for proteincontent in Fleckvieh and German Holstein, respectively. Conversely, negativeeffects of the lysine variant of -242 to -180 kg for Fleckvieh and -260 to -320kg for German Holstein were revealed for milk yield from first to thirdlactation, resulting in enhanced fat yield of 7.5 to 14.8 kg in Fleckvieh and7.6 to 10.7 kg in German Holstein. For protein yield, however, mainly negativeeffects of -3.6 to 0.2 kg in Fleckvieh and -4.8 to -5.2 kg in German Holsteinwere observed. Pearson correlations between residuals of milk yield and contenttraits were decreased when omitting DGAT1 effects in the analysis, therebyindicating that DGAT1 contributes to negative correlations between these traits.Molecular tests allow for the direct selection among variants; however, thebenefits of the alternative alleles depend on economic weights given to thedifferent milk production traits in the breeding goal.