SummaryThe free fatty acid (FFA) levels after milking (initial FFA) and after 22 h storage at 4°C (FFA-22) were determined in milk from 57 cows every 2 weeks for one year. FFA levels which were higher in summer than in winter were explained by later stages of lactation occurring in summer. Initial FFA content increased regularly during lactation. FFA-22 content increased after 12 weeks, and further after 32 weeks of lactation. The first increase was due to stage of lactation. The second increase, which appeared in late lactation, was essentially due to the stage of pregnancy combined with the effect of milk yield. FFA-22 content increased in the second part of pregnancy, chiefly after 24 weeks. A low milk yield (≼ 5 kg) further increased lipolysis, but only after 16 weeks of pregnancy. The effect of stage of pregnancy may have been enhanced by management factors such as feeding conditions. In this investigation, the seasonal FFA variation in bulk tank milk could be explained entirely by physiological factors.