Cationic polyelectrolytes (chitosan, poly-L-lysine (PLL), polyethyleneimine (PEI) and trimethylammonium glycol chitosan iodide (TGCI)) were used to entrap anoxygenic phototrophic bacteria in order to prevent the inhibitory effect of NH4+ on hydrogen production. When combined with agar gel, chitosan and PLL demonstrated no obvious repressive effect on hydrogen production by Rhodobacter sphaeroides under light-anaerobic conditions with lactate and glutamate as the carbon and nitrogen sources, respectively. On the other hand, both PEI and TGCI exerted a detrimental effect on hydrogen production under these conditions. Hydrogen production in the presence of NH4+ by the bacteria entrapped in the complex gel containing chitosan and agar improved considerably compared to that in the control containing only agar. Evidence shows that chitosan improves the hydrogen production via various effects. Diffusion tests demonstrated that the addition of chitosan increased to some extent the resistance to the diffusion of positively charged NH4+, but had no effect on negatively charged lactate. A buffer effect in the complex gels was also revealed.