Several lines of evidence have recently underscored the significance of fatty acids or fatty acid-derived metabolites as signaling molecules in adipocyte differentiation. The acyl-CoA-binding protein (ACBP), which functions as an intracellular acyl-CoA pool former and transporter, is induced during adipocyte differentiation. In this report we describe the effects of expression of high levels of ACBP antisense RNA on the differentiation of 3T3-L1 cells. Pools of 3T3-L1 cells transfected with vectors expressing ACBP antisense RNA showed significantly less lipid accumulation as compared with cells transfected with the control vector. When individual clones were analyzed the degree of differentiation at day 10 was inversely correlated with the level of ACBP antisense RNA expression at day 0. Furthermore, in the clones with the highest levels of ACBP antisense expression, the induction of expression of the adipogenic transcription factors peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha as well as several adipocyte-specific genes was significantly delayed and reduced. The adipogenic potential of antisense-expressing cells was partially restored by transfection with a vector expressing high levels of ACBP. Taken together, these results are strong evidence that inhibition of differentiation is causally related to the decreased expression of ACBP, indicating that ACBP plays an important role during adipocyte differentiation.