Rat liver fatty acid binding protein (L-FABP) and rat intestine fatty acid binding protein (I-FABP) are homologous proteins which are both found in intestinal epithelial cells. It was once well accepted that liver fatty acid binding protein bound fatty acyl-CoAs, but the recent finding of a novel acyl-CoA binding protein (ACBP) in preparations of L-FABP has challenged the role of FABPs in acyl-CoA metabolism. Prior to the discovery of ACBP, L-FABP preparations from liver were shown to modulate the rate of fatty acyl-CoA synthesis (Burrier et al., 1987) and their conversion to phospholipids (Bordewick et al., 1989). Studies using FABPs free of ACBP are needed to determine the role of I-FABP and L-FABP in fatty acyl-CoA metabolism. In this study, highly pure recombinant L-FABP and I-FABP were used first to establish binding to fatty acyl-CoAs and then to examine the effects of these FABPs on microsomal phosphatidic acid synthesis. The standard Lipidex-1000 binding assay using [14C]oleoyl-CoA and a new fluorescence binding assay using the fluorescent fatty acyl-CoA cis-parinaroyl-CoA were used to determine binding. The results of these assays indicate that L-FABP binds fatty acyl-CoAs at two sites with a high-affinity Kd = 3-14 microM. These binding assays showed that I-FABP has a much lower affinity for fatty acyl-CoAs than does L-FABP. Furthermore, in vitro only L-FABP significantly increases the rate of incorporation of oleoyl-CoA into lysophosphatidic acid and phosphatidic acid.