Activities of carboxylation enzymes were analyzed in the mycelium of the mycorrhizal fungus Amanita muscaria (L. ex Fr.) Hooker, in non-mycorrhizal short roots of Norway spruce (Picea abies [L.] Karst.) and in myconhizas of these two partners. While pyruvale carboxylase (PC, EC 18.104.22.168) and phosphoenolpyruvate carboxykinase activities (PEPCK.EC 22.214.171.124) could be detected in the mycelium of A. muscaria, phosphoenolpyruvate carboxyknase (PEPC, EC 126.96.36.199) was only active in root tissue. In A. muscaria, PC activity was generally low (around 10 nmol mg−tprotein min−) but PEPCK activity was above 250 nmol mg−1 protein min−1. Mycorrhizal development on short roots decreased PEPC activity by more than 75%, although dilution by the fungal biomass in mycorrhizas was only 35%. This reduction in activity was paralleled by a decreased content of PEPC protein. By means of micro-analytical methods it was shown that PEPC activity was lowest in the central zones of the mycorrhizas, Whereas PEPC activity was highest in the corresponding central sections in non-mycorrhizal short roots. 14CO2 labelling, on the other hand, revealed that in vivo CO2 fixation was higher in mycorrhizas compared to non-mycorrhizal short roots. It is concluded that fungal carboxylases (probably PEPCK) are important for anaplerotic CO2 fixation during nitrogen assimilation in mycorrhizas of Norway spruce.