A glucose utilizing mutant was selected from parent strain Alcaligenes eutrophus H16, and named as Glu-9. The mechanisms of glucose utilization of the mutant Glu-9 was investigated by measuring the D-[1-¹⁴C] glucose transport activity and the activities of key enzymes related to glucose and fructose uptake via facilitated diffusion. The uptaken glucose seems to activate key enzymes related to glucose matabolism. The selectivity between glucose and fructose of mutant Glu-9 was also analyzed by measuring glucose transport activity and enzyme activities under the various cultivation conditions using different carbon sources. Mutant Alcaligenes eutrophus Glu-9 preferentially consumed fructose from mixed substrates of glucose and fructose due to the inhibition of fructose to glucose transport activity. The characteristics of cell growth and PHB accumulation of Alcaligenes eutrophus Glu-9 were examined under various cultural conditions. Mutant strain Glu-9 showed tolerance in high concentration of glucose and increased yield of PHB production.