A cDNA encoding a novel fatty acid transport protein (FATP) was identified recently using expression cloning methodologies. We have studied the expression of FATP in differentiating 3T3-L1 cells and adipose tissue in vivo. When 3T3-L1 preadipocytes are treated with a combination of methylisobutylxanthine, dexamethasone, and insulin to induce differentiation, the abundance of FATP mRNA decreases within 24 h to less than one-third that of preadipocytes and increases subsequently, such that mature adipocytes have 5-7 times more FATP mRNA than fibroblastic precursors. In fully differentiated 3T3-L1 adipocytes, insulin alone is sufficient to down- regulate FATP mRNA levels 10-fold. The concentration of insulin necessary for half-maximal repression (I0.5) is ~1 nM and is specific for insulin; insulin-like growth factor I (IGF-I) has little effect at similar concentrations. Kinetic analysis indicates that the reduction in expression of FATP mRNA by insulin is rapid (t 1/4 = ~4h) and reversible upon withdrawal of insulin. The half-lives of FATP mRNA are 2.9 h and 4.4 h in the absence and presence of insulin, respectively. The insulin-mediated decrease in FATP steady state mRNA level correlates with a decrease in its transcription rate as measured by nuclear run-on transcription assay. To determine whether physiological conditions that alter insulin concentration in vivo affect adipose FATP levels, feeding/fasting studies are employed. Fasting of C57BL/6J mice for 48 h results in an 11-fold up-regulation of FATP mRNA expression in adipose tissue. Refeeding of fasted animals for 72 h results in a return of FATP mRNA to basal levels. In sum, these results indicate that the expression of FATP gene is negatively regulated by insulin at the transcriptional level in cultured adipocytes and that transporter mRNA expression in murine adipose tissue is altered in a manner consistent with insulin being a negative regulator of gene activity.