Biohydrogen is an ideal energy carrier for mobile chemical fuel cells, but its use is often limited by unavailability of sustained H(2) production system(s). Here, we developed a compact system for H(2) production from formate based on immobilized cells of recombinant Escherichia coli SH5. Three different matrices were tested as immobilization medium, among which agar showed the best performance in mechanical stability and permeability of substrate(s) and/or gaseous products (H(2) and CO(2)). To explore and optimize the H(2) production capability of the immobilized cells, the conditions for cell immobilization including cell loading and agar concentration as well as the factors affecting H(2) production rate such as temperature, pH, and substrate concentration were studied in detail. A maximum volumetric production rate of 2.4 L H(2) L(-1) h(-1) was obtained when the immobilized cells were incubated with 350 mM sodium formate at pH 6.5 and 37 degrees C. Periodic supplementation of 200 mM formate with 20 mM glucose at pH 6.5 maintained the high H(2) production rate for a prolonged period of 10 h. We believe that our process can be developed for sustained H(2) production and is applicable to the operation of fuel cells in small-scale. Copyright (C) 2010, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.