A photoaffinity labeling method was developed to identify and characterize high affinity fatty acid-binding proteins in membranes. The specific labeling of these sites requires the use of low concentrations (nanomolar) of the photoreactive fatty acid 11-m-diazirinophenoxy-[11-3H]undecanoate. It was delivered as a bovine serum albumin (BSA) complex which serves as a reservoir for fatty acid and thus allows precise control of unbound fatty acid concentrations. The fadL protein of E. coli, which is required for fatty acid permeation of its outer membrane, was labeled by the photoreactive fatty acid neither specifically nor saturably when the probe was added in the absence of BSA; however when a nanomolar concentration of the uncomplexed probe was maintained in the presence of BSA, the labeling of the fadL protein was highly specific and saturable. This photoaffinity labeling method was also used to characterize a 22 kDa, high affinity fatty acid-binding protein which we have recently identified in the plasma membrane of 3T3-L1 adipocytes. This protein bound the probe with a Kd of 216 nM. The approach described is easily capable of identifying membrane-bound fatty acid-binding proteins and can distinguish between those of high and low affinities for fatty acids. It represents a general method for the identification and characterization of fatty acid-binding proteins.