The pathways of glycerol-3-P (G3P) generation were examined in retroperitoneal (RETRO) and epididymal (EPI) adipose tissues from rats fed a cafeteria diet for 3 wk. The cafeteria diet induced marked increases in body fat mass and in the plasma levels of insulin and triacylglycerol (TAG). RETRO and EPI from cafeteria diet-fed rats had increased rates of norepinephrine turnover (143 and 60%, respectively) and of de novo fatty acid (FA) synthesis (58 and 98%), compared with controls fed a balanced commercial diet. Cafeteria diet feeding induced marked increases in RETRO and EPI in vivo rates of glucose uptake (52 and 51%, respectively), used to evaluate G3P generation via glycolysis, as well as in glycerokinase activity (119 and 36%) and TAG-glycerol synthesis from glycerol (56 and 71%, respectively). In contrast, there was a marked reduction of glyceroneogenesis in RETRO and EPI from cafeteria diet-fed rats, which was evidenced by the significant decreases of P-enolpyruvate carboxykinase (PEPCK-C) activity (48 and 36%) and TAG-glycerol synthesis from pyruvate (45 and 56%, respectively). Denervation of RETRO from cafeteria diet-fed rats reduced the activity of glycerokinase by 50%, but did not affect glucose uptake or PEPCK-C activity and TAG-glycerol synthesis from pyruvate by the tissue. The data show that glyceroneogenesis can also be inhibited to adjust the supply of G3P to the existing rates of FA esterification and TAG synthesis and suggest that this adjustment is made by reciprocal changes in the generation of G3P from glucose via glycolysis and from glyceroneogenesis, independently from G3P production by glycerokinase.