Evolution of 14CO2 from onion roots and the intraradical hyphae of Gigaspora margarita Becker & Hall was examined by radiorespirometry after addition of 14C-labelled glucose or sucrose to mycorrhizal or non-mycorrhizal roots. In mycorrhizas, the respiration rate from glucose was about twice that from sucrose. The respiration rate from glucose in the mycorrhizas was much higher than that in the non-mycorrhizal roots, but no differences between mycorrhizal and non-mycorrhizal roots were found in the respiration from sucrose. Intraradical hyphae were isolated from mycorrhizas by enzyme digestion and homogenization followed by Percoll® gradient centrifugation. The 14C-labelled glucose, fructose or sucrose was added to the isolated hyphae and the subsequent evolution of 14CO2 was measured. The hyphae mainly used glucose as a substrate for respiration. Although sucrose or fructose was utilized to some degree, the respiration rate from glucose was much higher than that from sucrose and fructose. This is the first direct evidence of use of glucose by the intraradical hyphae of arbuscular mycorrhizal fungi in the symbiotic state.