A multi-layered photobioreactor (MLPR), where the light paths were formed by the localization of bacterial cells, was constructed for efficient hydrogen production. The performance was investigated under several conditions in order to clarify the effect of this reactor on hydrogen production. An analysis of the hydrogen production profile showed that the MPLR utilizes both the light that directly illuminates its surface and the light induced and diffused from its light paths for hydrogen production. It was also found that the hydrogen productivity in the MLPR was more than twice that in a plate-type reactor. When a photosynthetic bacterium mutant with reduced pigment, MTP4, was used, the maximum hydrogen production rate reached 2.01/m(2)h, which was 38% higher than that of a conventional plate-type reactor. The synergistic effect of the improvement in the reactor and the modification of the bacteria was brought about by the combination of the MLPR and MTP4, and resulted in an improvement in the hydrogen production. (c) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.