A series of noncovalent complexes formed between the 86 residue acyl CoA binding protein (ACBP) and a series of acyl CoA derivatives has been studied by electrospray ionization mass spectrometry. Conditions were found under which CoA ligands can be observed in the mass spectrometer bound to ACBP. Despite the very low dissociation constants (10-7 to 10-10 M) of the acyl CoA ligand complexes high ratios of ligand-to-protein concentration in the electrospray solution were found to increase the proportion of intact complex observed in the spectrum. Variation in the length of the hydrophobic acyl chain of the ligand (C16, C12, C8, C0) resulted in similar proportions of complex observed in the mass spectrum even though significant variation in solution dissociation constants has been measured. A substantially reduced proportion of complex was, however, found for the mutant proteins, Y28N, Y31N, and Y73F, lacking tyrosine residues involved in critical interactions with the CoA ligand. These results have been int...