Milk spontaneous lipolysis (SL) of milk triglycerides is induced by the lipoprotein lipase, a milk native enzyme, and leads to an accumulation of partial glycerides and free fatty acids that are responsible for the deterioration of the taste of milk products. The gene coding for diacylglycerol acyltransferase 1 (DGAT1), an enzyme implicated in triglycerides synthesis, has an important polymorphic site at the K232A locus. This gene is well known to modulate milk composition. No data are available on the effects of DGAT1 on SL. Thus, a trial was carried out to evaluate the effects of DGAT1 K232A polymorphism on milk SL upon milking frequency variations [once- (ODM) and twice-daily milking (TDM)]. Twenty-one cows were divided into 3 groups according their DGAT1 K232A genotype: 8 cows had the KK genotype of DGAT1 (KK cows), 8 had the KA genotype (KA cows), and 5 had the AA genotype (AA cows). The trial consisted in 3 successive periods: 3 wk of TDM, 3 of ODM, and 4 of TDM. Samples were collected for fat and protein contents, SL, fatty acid, and protein profiles determinations. The KK cows presented higher fat and protein contents, lower milk production, and higher κ-casein percentage. No significant difference in fatty acid composition was noted between groups. The SL was twice as high for KK cows in TDM situations (1.13 vs. 0.59 and 0.63 mEq/100 g of fat, respectively, for KK, KA, and AA cows during the first period of TDM, and 0.46 vs. 0.25 and 0.21 mEq/100 g of fat, respectively, for KK, KA, and AA during the second period of TDM). The SL remained sharply lower in TDM2 than in TDM1. During ODM, no difference in SL was found between groups and SL remained below 0.2 mEq/100 g of fat. These results demonstrate the existence of a correlation between DGAT1 genotypes and spontaneous lipolysis, in interaction with an environmental factor, milking frequency, although it has not been possible to clarify the causal mechanism at this stage.