In the present investigation, we have compared the potential of triacylglycerol formation from sn-glycerol-3-phosphate (GP) and 2-monoacylglycerol (MG) in liver, adipose tissue and intestine from lean and obese Zucker rats. Microsomal fractions were used to measure the sn-glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase (DGAT) and monoacylglycerol acyltransferase (MGAT) activities and homogenates were used to measure NEM-sensitive and NEM-insensitive phosphatidate phosphohydrolase (PPH) activities. In adipose tissue and liver, the GP pathway served as the major route of glycerolipid formation, with adipose tissue being 5-20-fold more active. The activities of the GP pathway enzymes increased further in response to obesity, with some degree of organ specificity. In adipose tissue of obese rats, the activities of all the pathway enzymes increased; whereas, in liver and intestine, this response was limited to PPH and GPAT, respectively. In contrast with the GP pathway enzymes, obesity in Zucker rats was not associated with alterations in the acylation of 2-monoacylglycerol. Comparison of the activities of MGAT in different intestinal segments indicated that the MG pathway was most active in the jejunum and least active in the ileum and that this pattern did not change in response to obesity. These measurements of the individual enzyme reactions provide evidence that the entire process of esterification via sn-glycerol-3-phosphate is accelerated in the various organs from obese rats and that this perturbation in lipid metabolism may contribute significantly to the increased deposition of body fat noted in this animal model.