Interaction of rat and human cystathionine-beta -synthase (CBS) with various potential ligands has been studied by visible and EPR spectroscopy in order to explore the coordination chemistry of this atypical hemeprotein, Ferric CBS did not react with any classical hemeprotein ligands, such as various imidazole and pyridine derivatives, N-3(-) and isonitriles RNC. Ferrous CBS also failed to bind these nitrogenous ligands or nitrosoalkanes. However, it reacts with various isonitriles RNC, leading to complexes characterized by a Soret peak at 433 +/- 2 nm. Binding of isonitriles to ferrous CBS is a relatively slow process; its rate markedly depends on the nature of R. It thus seems that the only exogenous ligands able to bind CBS iron are carbon-centered, very strong heme-Fe(II) ligands such as CNR, CO, and CN-, presumably after dissociation of the CBS-iron(II)-cysteinate bond. Isonitriles appear as interesting tools for further studies on the topology of CBS active site.