A L-lactate-selective microbial biosensor was developed using permeabilized cells of gene-engineered thermotolerant methylotrophic yeast Hansenula polymorpha, over-producing L-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, flavocytochrome b(2), FC b(2)). The construction of FC b(2)-producers by over-expression of the gene CYB2 H. polymorpha encoding FC b(2) is described. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in the frame of a plasmid for multicopy integration was transformed to the recipient strain H. polymorpha C-105 (gcr1 catX) impaired in glucose repression and devoid of catalase activity. The permeabilized cells were either immobilized on the graphite working electrode by physical entrapment of the cell suspension by means of a dialysis membrane or by integration of the cells in an electrochemically generated layer using a cathodic electrodeposition polymer. Phenazine methosulphate was used as a free-diffusing redox mediator. It was assumed that the mediator reacts with mitochondrial FC b(2) after entering the cells in the presence of L-lactate. The biosensor based on recombinant yeast cells exhibited a higher K(M)(app) value and hence expanded linear range toward L-lactate as compared to a similar sensor based on the initial cells of H. polymorpha C-105.