Acyl-coenzyme A binding protein from bovine liver and the protein expressed in Escherichia coli by the recombinant gene of this protein have been studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. This protein has, in addition to the ability to bind acyl-coenzyme A, been reported to have several important physiological and biochemical functions. It is known as the diazepam binding inhibitor, as a putative neurotransmitter, as a regulator of insulin release from pancreatic cells, and as a mediator in corticotropin-dependent adrenal steroidogenesis. The only difference between the protein produced by recombinant techniques and the native acyl-coenzyme A binding protein is the N-terminal acetyl group present only in the native protein. The two proteins have 86 amino acid residues and a molecular mass of approximately 10,000 Da. Complete assignment of the 1H nuclear magnetic resonances has been obtained for a major proportion of the amino acid residues (55 residues), and partial assignment has been achieved for the others (31 residues). Sequential nuclear Overhauser effects have demonstrated that the protein has a secondary structure consisting of four alpha-helices of residues 1-15, 22-35, 52-60, and 68-85. Furthermore, a large number of long-range nuclear Overhauser effects have been identified, indicating that the assignment given here will provide a basis for a structure determination of this protein in solution by nuclear magnetic resonance spectroscopy.