Pyruvate synthase (pyruvate:ferredoxin oxidoreductase, PFOR) catalyzes the interconversion of acetyl-CoA and pyruvate, but the reductive carboxylation activities of heterotetrameric PFORs remain largely unknown. In this study, we cloned, expressed, and purified selected six heterotetrameric PFORs from hyperthermophilic archaea. The reductive carboxylation activities of these heterotetrameric PFORs were measured at 70?°C and the ratio of reductive carboxylation activity to oxidative decarboxylation activity (red/ox ratio) were calculated. Four out of six showed reductive decarboxylation activities. Among them, the PFOR from Pyrolobus fumarii showed the highest reductive carboxylation activities and the highest red/ox ratio, which were 54.32 mU/mg and 0.51, respectively. The divergence of the reductive carboxylation activities and the red/ox ratios of heterotetrameric PFORs in hyperthermophilic archaea indicate the diversity of the functions of PFOR over long-term evolution. This can help us better understand the autotrophic CO fixation process in thermal vent, or in other CO-rich high temperature habitat.