Dark chilling inhibited photosynthesis in two soybean [Glycine max (L.) Merr.] cultivars (Fiskeby V and Maple Arrow). The inhibition of CO₂ assimilation was characterized by a simultaneous decrease in stomatal conductance (G_s) and intercellular CO₂ concentration (C_i) in Maple Arrow, whereas a similar decrease in G_s in Fiskeby V occurred without any change in C_i. Dark chilling had little effect on total ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity, Rubisco protein content or Rubisco activation state in the subsequent light period. Chilling reduced the abundance of the nocturnal Rubisco inhibitor, 2-carboxyarabinitol 1-phosphate, only in Fiskeby V. The abundance of Rubisco small subunit transcripts was enhanced in both cultivars as a result of dark chilling. Dark chilling decreased the maximal extractable activities and activation states of stromal fructose-1,6-bisphosphatase (FBPase) and NADP-malate dehydrogenase but had no effect on sucrose phosphate synthase or leaf sucrose and starch contents. It is concluded that dark chilling-induced limitations on CO₂ assimilation are predominantly due to metabolic restrictions rather than to direct effects on electron transport reactions and that stromal FBPase is particularly susceptible to dark chilling.