The magnitude and role of dark CO(2) fixation were examined in nodules of intact soybean plants (Harosoy 63 x Rhizobium japonicum strain USDA 16). The estimated rate of nodule dark CO(2) fixation, based on a 2 minute pulse-feed with (14)CO(2) under saturating conditions, was 102 micromoles per gram dry weight per hour. This was equivalent to 14% of net nodule respiration. Only 18% of this CO(2) fixation was estimated to be required for organic and amino acid synthesis for growth and export processes. The major portion (75-92%) of fixed label was released as CO(2) within 60 minutes. The labeling pattern during pulse-chase experiments was consistent with CO(2) fixation by phosphoenolpyruvate carboxylase. During the chase, the greatest loss of label occurred in organic acids. Exposure of nodulated roots to Ar:O(2) (80:20) did not affect dark CO(2) fixation, while exposure to O(2):CO(2) (95:5) resulted in 54% inhibition. From these results, it was concluded that at least 66% of dark CO(2) fixation in soybean may be involved with the production of organic acids, which when oxidized would be capable of providing at least 48% of the requirement for ATP equivalents to support nitrogenase activity.