The method of continuous variation (Job plot analysis) and difference absorbance spectroscopy were used to investigate the binding of 2'(3')-(trinitrophenyl)-ADP and -ATP to chloroplast coupling factor 1 (CF1). Experiments performed at a low total concentration (30 microM) of nucleotide and enzyme binding sites (assuming three or four binding sites per CF1) could be interpreted in terms of approximately three nucleotide binding sites per CF1. At higher total concentrations (100 and 400 microM), the number of apparent binding sites increased to almost four. Computer-generated Job plots, using a protein-ligand complex formation scheme of n independent, nonequivalent binding sites, gave good fits to the experimental data at all concentrations when four binding sites were modeled. The dissociation constant of the fourth site was estimated to be approximately 20 microM. Additional nucleotide binding sites were not directly observed by this method and, if they exist, have very weak binding affinities (dissociation constants greater than approximately 1 mM).