Abstract The interaction of firefly luciferase with several triazine dyes has been investigated. Of those tested, the anthraquinone dye, Procion blue MX-R, displayed relatively high affinity [dissociation constant ( K D) 5μ M] and irreversibly inactivated the enzyme in a time dependent fashion. The substrates, MgATP (ATP is adenosine triphosphate) and luciferin, protected the enzyme against inactivation with Procion blue MX-R and following quantitative inactivation, incorporation of 1 mol dye per mol enzyme [molecular weight ( M r) 50 000] was observed. It is suggested that Procion blue MX-R binding is active site-directed and that the dye binding traverses both MgATP and luciferin binding sites. These data are exploited to evolve an improved purification protocol for firefly luciferase.