After growth on various carbon sources, sonic extracts of Hydrogenomonas facilis contained ribulosediphosphate (RuDP) carboxylase and phosphoribulokinase (Ru5-P kinase). After very short sonic treatment, a reductive adenosine triphosphate (ATP)-dependent incorporation of (14)CO(2) was also detectable. Reduced nicotinamide adenine dinucleotide (NADH(2)) served as reductant 30-fold more effectively than reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)). Adenosine 5'-phosphate (AMP) and adenosine 5'-pyrophosphate (ADP) inhibited Ru5-P kinase and NADH(2)-, ATP-dependent CO(2) fixation. The levels and duration of CO(2) fixation suggested that it is a cyclic process. The requirement of reduced pyridine nucleotide and ATP and the sensitivity of fixation to AMP and ADP support the conjecture that it occurs via the Calvin cycle. After thorough study of variables affecting catalysis, specific activities (millimicromoles of substrate disappearing per milligram of protein) at 30 C were determined for RuDP carboxylase (C), Ru5-P kinase (K) and ATP-, NADH(2)- dependent CO(2) fixation (CO(2) F) after growth autotrophically on fructose, glucose, ribose, glutamate, lactate, succinate, and acetate. Values for these growth modes were, respectively-for C: 67.3, 51.1, 51.4, 24.6, 2.05, 10.2, 2.25, 1.4; for K: 24.7, 24.0, 23.2, 14.2, 12.8, 12.9, 13.4, 2.8; and for CO(2) F: 4.54, 4.83, 3.10, 2.87, 0.85, 1.51, 0.24, 0.41. The qualitative parallel between values for RuDP carboxylase and CO(2) fixation suggests that one major control point in fixation is the step catalyzed by RuDP carboxylase.