Spontaneous lipolysis is the result of the activity of a native milk enzyme, the lipoprotein lipase. Lipolysis leads to a release of free fatty acids (FFA) that cause rancid flavor in milk products. A trial was carried out to study the effects of breed, energy levels, and lactation stage on milk spontaneous lipolysis in dairy cows. Sixty-four cows were used through a year of lactation. Cows were divided into 4 homogenous groups according to their breed [Holstein (HO) or Normande (NO)] and to their feeding system: the intensive system, with a high energy diet (HED) (in winter, corn silage with 30% concentrate; otherwise, pasture with 4 kg/d of concentrate) and the grass system (GS) (in winter, conserved grass with no concentrate; otherwise, pasture with no concentrate). The period of calving was synchronized between January and March. Individual milk samples were collected every month from both morning and evening milkings. The FFA levels were determined by the difference between the FFA levels after milking and the FFA after 24 h of storage at 4°C. Data were analyzed using SAS mixed procedure. We showed that FFA levels were higher in the evening milks (0.45 vs. 0.25 mEq/100 g of fat, P < 0.001) and that evening milks were more susceptible to lipolysis variations. HO cows were more susceptible to lipolysis than NO (0.34 vs. 0.20 mEq/100 g of fat, in morning milks; 0.62 vs. 0.33 mEq/100 g of fat in evenings milks, P < 0.001). There was no effect of the feeding system on morning milks lipolysis but GS had a tendency to increase milk lipolysis in evening milks (0.41 vs. 0.55 mEq/100 g of fat, P = 0.06). Lipolysis was higher in early and late lactation stage, particularly in the GS group. Conserved grass may also affect the lipolysis rate. The energy status in early and late lactation stage is frequently negative so the cows may be more susceptible to a low energy diet. No correlation was found between lipolysis and milk fat content. Poor correlations (r < 0.4, P < 0.001) were found between lipolysis and milk production, fat globule size, proportion of fatty acid and protein composition, body condition and weight.