Cell suspensions of uptake-hydrogenase-deficient (hup(-)) mutants of a wild-type (B10S) and a nifHDK deletion strain of Rhodobacter capsulatus were used comparatively to characterize the conventional, Mo-containing and the alternative, "iron-only" nitrogenase of this organism by determining the H-2 production and acetylene reduction activities under argon and dinitrogen atmospheres. A comparison with the corresponding hup(+) strains revealed that the hup(-) mutation did not affect the nitrogenase activity and specificity within the acetylene-reduction assay, but caused a significant increase in H-2 production, which was more prominent in the case of the Delta nifHDK strain. The Delta nifHDK hup(-) cells, grown in Mo-depleted medium and, thus, expressing the alternative nitrogenase system, were more than ten times less active in the acetylene-reduction assay but exhibited H-2 production rates equivalent to about 60% of the rates obtained with B10S hup(-) cells after growth in a medium containing 10 mu M MoO4-. When these conditions were applied, the BIOS strain only expressed the Mo nitrogenase. Under an argon atmosphere containing about 5.5% (v/v) acetylene and under a dinitrogen atmosphere, Delta nifHDK hup(-) cells produced H-2 at even higher rates than B10S hup(-) cells. The implications of our findings on a possible biotechnological H-2 production and on the mechanism of nitrogenase catalysis are considered.