A laboratory-made spectroluminometer was used to analyse the light emitted by firefly (Photinus pyralis) luciferase reacting with several nucleotide derivatives. The analysis of the light emission in the presence of ATP or dATP provides some evidence that the enzyme has two nucleotide binding sites, each one leading to the formation of a complex emitting mainly at 575 nm (ATP) or 610 nm (dATP). AMP is able to displace dATP from the second site (610 nm) to the first one. Photoaffinity labelling of the second site by 8-azido-AMP gives similar results. The amplification effect of CoA and acetyl-CoA is also reconsidered according to this model.