The activity of alpha-ketoglutarate dehydrogenase (KGDH) was assayed in cell-free yeast homogenates with respect to studying the mechanism of alpha-ketoglutarate (KG) overproduction by the thiamine-heterotrophic culture of Candida lipolytica in media with hexadecane. Two types of yeast cells were used for analysis: (1) cells grown in the conditions of thiamine excess (500 mcg/l) and taken at the logariphmic growth phase; (2) cells grown in the conditions of thiamine deficiency and taken at the beginning of KG overproduction when the cultural growth was limited by thiamine. The activity of KGDH was assayed in the absence of thiamine diphosphate (TDP) and in its presence, the content of the holoenzyme and the total content of the holoenzyme and the apoenzyme being determined respectively. The activity of holoKGDH in the thiamine-deficient cells of C. lipolytica producing KG was almost five times lower than in the cells grown in the conditions of thiamine excess. In the latter, all KGDH was in the form of the holoenzyme; in the thiamine-deficient cells, 90% of KDGH was in the form of the apoenzyme devoid of TDP and therefore lacking the enzyme activity. The total content of holoKGDH IN THE THIAMINE-DEFICIENt cells producing KG was twice as high as in the cells grown in the conditions of thiamine excess. The data obtained are discussed in relation with the mechanism of KG biosynthesis from n-alkanes by yeast cells.