In order to determine whether critical enzyme activities of glycerolipid synthesis change seasonally in the golden-mantled ground squirrel (Spermophilus lateralis), we collected summer and winter samples of liver, brown adipose tissue (BAT), and white adipose tissue (WAT). Compared with fatty acid synthase activity during hibernation, summer activities were 2.5- to 8-fold higher in adipose tissue and liver. Diacylglycerol acyltransferase (DGAT) activity was 2.6-fold higher in WAT during the summer, consistent with increased seasonal triacylglycerol storage, but the activity did not change in liver or BAT, suggesting that in these tissues, triacylglycerol synthesis is equally active in summer and winter. Lack of change in acyl-CoA synthetase in liver and BAT may reflect high synthetic rates for acyl-CoAs that are destined in the summer for glycerolipid synthesis and in the winter for beta-oxidation. Monoacylglycerol acyltransferase (MGAT) activity increased significantly in both liver and WAT during the summer but decreased in BAT. Although the changes were consistent with active year-round triacylglycerol synthesis, the higher summer MGAT activity observed in the squirrel liver and WAT suggest that MGATs function may not be limited to conserving essential fatty acids during physiological states of lipolysis. Seasonal changes observed in the ground squirrel were similar to those previously reported in the yellow-bellied marmot (Marmota flaviventris), confirming that important adjustments occur in energy metabolism necessitated by long seasonal hibernation.