The liver-type fatty acid binding protein (L-FABP), a member of a family of mostly cytosolic 14-15 kDa proteins known to bind fatty acids in vitro and in vivo, is discussed to play a role in fatty acid uptake. Cells of the hepatoma HepG2 cell line endogenously express this protein to approximately 0.2% of cytosolic proteins and served as a model to study the effect of L-FABP on fatty acid uptake, by manipulating L-FABP expression in two approaches. First, L-FABP content was more than doubled upon treating the cells with the potent peroxisome proliferators bezafibrate and Wy14,643 and incubation of these cells with [1-14C]oleic acid led to an increase in fatty acid uptake rate from 0.55 to 0.74 and 0.98 nmol/min per mg protein, respectively. In the second approach L-FABP expression was reduced by stable transfection with antisense L-FABP mRNA yielding seven clones with L-FABP contents ranging from 0.03% to 0.14% of cytosolic proteins. This reduction to one sixth of normal L-FABP content reduced the rate of [1-14C]oleic acid uptake from 0.55 to 0. 19 nmol/min per mg protein, i.e., by 66%. The analysis of peroxisome proliferator-treated cells and L-FABP mRNA antisense clones revealed a direct correlation between L-FABP content and fatty acid uptake.