The metabolic pathway of gluconate, a major product of glucose metabolism during spore germination, was investigated in Bacillus megaterium QM B1551. Compared to the parent, mutant spores lacking gluconokinase could not metabolize gluconate, whereas the revertant simultaneously restored the enzyme activity and the ability to metabolize it, indicating that gluconokinase was solely responsible for the onset of gluconate metabolism. To identify a further metabolic route for gluconate, we determined 14C yields in acetate and CO2 formed from [14C]gluconate, and found that experimental ratios of 14CO2/[14C]acetate obtained from [2-14C]gluconate and [3,4-14C]gluconate were not compatible with the ratios predicted from the Entner-Doudoroff pathway. In contrast, when CO2 release caused by recycling (approx. 30%) was corrected, the ratios almost agreed with those from the pentose cycle. Comparison of specific radioactivities in acetate also supported the conclusion that gluconate was metabolized via the pentose cycle, subsequently metabolized via the Embden-Meyerhof pathway, and finally degraded to acetate and CO2 without a contribution by the Krebs cycle.