When leaf discs from spinach plants (Spinacia oleracea L.) maintained in the dark for several days were subsequently illuminated, the decrease of incorporated (14)CO2 measured under steady state conditions was found to be accompanied by an altered fixation pattern. Substances found to contain a significantly lower label, were malate and aspartate. In contrast, an enhanced incorporation of radioactivity was observed for those substances known to be formed during light respiration. Since the same tendencies were obtained at higher CO2 concentrations and after the removal of the lower epidermis, a new metabolic situation rather than an impaired CO2 supply, was considered to be responsible for the altered turnover of intermediates. The constant ratio in the labelling of intermediates formed in the chloroplasts and the known localization of enzymes involved in the formation of C-4 components led us to conclude that primarily the activity of cytoplasmic enzymes is influenced by dark.