The hyperlipidemia associated with obesity and type 2 diabetes is caused by an increase in hepatic triglyceride synthesis and secretion that is secondary to an increase in de novo lipogenesis, a decrease in fatty acid (FA) oxidation, and an increase in the flux of peripherally derived FA to the liver. The uptake of FA across the plasma membrane may be mediated by three distinct proteins--FA translocase (FAT), plasma membrane FA binding protein (FABP-pm), and FA transport protein (FATP)--that have recently been characterized. Acyl-CoA synthetase (ACS) enhances the uptake of FAs by catalyzing their activation to acyl-CoA esters for subsequent use in anabolic or catabolic pathways. In this study, we examine the mRNA levels of FAT, FABP-pm, FATP, and ACS in the liver and adipose tissue of genetically obese (ob/ob) mice and their control littermates. FAT mRNA levels were 15-fold higher in liver and 60-80% higher in adipose tissue of ob/ob mice. FABP-pm mRNA levels were twofold higher in liver and 50% higher in adipose tissue of ob/ob mice. FATP mRNA levels were not increased in liver or adipose tissue. ACS mRNA levels were higher in adipose tissue but remained unchanged in liver. However, the distribution of ACS activity associated with mitochondria and microsomes in liver was altered in ob/ob mice. In control littermates, 61% of ACS activity was associated with mitochondria and 39% with microsomes, whereas in ob/ob mice 34% of ACS activity was associated with mitochondria and 66% with microsomes; this distribution would make more FA available for esterification, rather than oxidation, in ob/ob mouse liver. Taken together, our results suggest that the upregulation of FAT and FABP-pm mRNAs may increase the uptake of FA in adipose tissue and liver in ob/ob mice, which, coupled with an increase in microsomal ACS activity in liver, will enhance the esterification of FA and support the increased triglyceride synthesis and VLDL production that characterizes obesity and type 2 diabetes.