Abstract The in vitro rate of esterification of fatty acids in adipose tissue was compared between cows that were fed at restricted energy intake and cows that were overfed during the dry period. Subcutaneous adipose tissue was biopsied at –1, 0.5, 1, and 3 wk from parturition. The basal in vitro rate of esterification was quantified, as well as the rate of esterification after the addition of glucose or glucose plus insulin. The basal rate in adipose tissue from overfed cows at –1 wk was higher than in adipose tissue from cows that were fed at restricted energy intake and indicated enhanced storage of triacylglycerols in adipose tissue of overfed cows at that time. The rate of esterification after the addition of glucose or glucose plus insulin was increased in both groups at each sampling time, but the mean rates, expressed as a percentage of the basal rates, were lower for overfed cows than for cows that were fed at restricted energy intake at 0.5 and 1 wk. Although the addition of glucose or glucose plus insulin increased esterification rates in adipose tissue from both groups of cows, adipose tissue from overfed cows was less sensitive to the addition of these compounds. In conclusion, overfeeding during the dry period predisposed cows to accumulate fat in adipose tissue during the prepartum period. The smaller increase in esterification rate after the addition of glucose or glucose plus insulin in adipose tissue of overfed cows indicates a lower ability of the adipose tissue to esterify circulating fatty acids or to reesterify mobilized fatty acids, which, combined with higher rates of lipolysis postpartum, contributes to continuously elevated concentrations of circulating nonesterified fatty acids postpartum, leading to a more severe hepatic lipidosis in overfed cows.