Alkyl-substituted Sepharose 4B prepared by the glycidyl ether method with different degrees of substitution and alkyl chain length, have been used as a non-ionic matrix for immobilization of bacterial luciferases. Exposure of hydrophobic clusters in the protein molecule was confirmed by fluorescence studies using 8-anilino-1-napthalene-sulfonate as a hydrophobic reporter. Immobilization of luciferases took place with high efficiency and retention of their basic kinetic properties including a high recovery of activity, which was dependent on the alpha and not the p subunit of the heterodimeric enzyme. A remarkable increase in thermal stability was shown for Vibrio harveyi (Vh) luciferase on binding to octyl-Sepharose. As FMN reductase was coimmobilized with Vh luciferase, the reducing power for the reaction (FMNH2) could also be supplied by NAD(P)H. The catalytic potential of the coimmobilized enzymes was dramatically improved apparently due to reduction of FMNH2 auto-oxidation, as reported earlier. The potential application of this method to measure different compounds that can reduce NAD(P) is thereby increased. The simplicity of adsorption of luciferase on these matrices together with the high activity, reusability and increased thermal stability suggests that the method of immobilization described may provide a useful procedure for bioassays when coupled to the bacterial luminescence reaction.