Fatty acid-binding protein (FABP) was identified and isolated from rat liver cytosol by gel filtration, thin layer isoelectric focusing, and affinity chromatography. FABP (Mr 12,080 +/- 80) exists in several immunochemically identical forms differing in isoelectric pH, which may in part reflect differences in their respective complements of bound endogenous ligand. FABP-bound fatty acids accounted for 60% of total cytosolic long chain fatty acids but contained no detectable phospholipid; the substantial enrichment of FABP in 18:2 and 20:4 as compared with whole liver homogenate was not influenced by homogenization of tissue in EDTA. The amino acid composition of FABP suggests that it is closely related or identical with certain similar neutral and acidic cytosolic proteins reported from other laboratories. By quantitative radial immunodiffusion, FABP concentration in cytosol from livers of sexually mature female rats exceeded that from mature males (51.7 +/- 3.0 versus 39.8 +/- 4.0 micrograms/mg of protein, p less than 0.05), confirming earlier studies in which sex steroid effects on rates of fatty acid utilization were correlated with FABP concentration as determined by means of a binding assay. The abundance of FABP, its importance in the cytosolic binding of endogenous as well as exogenous fatty acids, and its demonstrated correlation with rates of hepatocyte fatty acid utilization provide additional evidence for its relationship to the cellular metabolism of long chain fatty acids.