A two stage culture was examined as a new method for the cultivation of Alcaligenes eutrophus to produce poly-D-3-hydroxybutyric acid, P(3HB), from H-2, O-2 and CO2 without danger of gas explosion. In this culture method, the microorganism was grown beforehand in an organic medium under heterotrophic conditions for exponential growth, and the cells were then cultivated for P(3HB) accumulation under autotrophic conditions in which the O-2 concentration in the substrate gas was below the explosion limit of 6.9%. Although the K-L alpha of the fermenter used was not high (ca. 340 h(-1)), it was possible to obtain P(3HB) at a relatively high production rate (0.56-0.91 g.dm(-3).h(-1)) and concentration (15.23-23.91 g dm(-3)) in the autotrophic stage, even under such a low O-2 concentration.