d(-) Lactate oxidation in Azobacter vinelandii strain O is readily carried out by the membrane bound enzyme that concentrates in the electron transport fraction (R(3)). This oxidation in the R(3) fraction is not dependent on externally added nicotinamide adenine dinucleotide, flavine adenine dinucleotide, or flavine mononucleotide. Phenazine methosulfate, O(2), and menadione all served as good electron carriers, and the oxidation of lactate was limited to the d(-) stereoisomer. Of all the alpha-hydroxyacids examined, only d(-) lactate and d(-) alpha-hydroxybutyrate were oxidized by the R(3) fraction. Paper chromatographic studies revealed that the 2,4-dinitrophenylhydrazine derivative formed from d(-) lactate oxidation was pyruvate. Pyruvate, in turn, could be further decarboxylated nonoxidatively by the R(3) fraction. Spectral studies revealed that both the R(3) flavoprotein and cytochrome (a(2), a(1), b(1), c(4), and c(5)) components were reduced by d(-) lactate. The d(-) lactic oxidase activity was sensitive to electron transport inhibitors, i.e., chlorpromazine, antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide, rotenone, dicumarol, and cyanide, and to a small extent to 4,4,4-trifluoro-1-(2-thienyl)-1,3-butane-dione (TFTB) and Amytal. The d(-) lactic phenazine methosulfate and menadione reductases were sensitive only to dicumarol and TFTB. Chlorpromazine was found to be a highly specific inhibitor of d(-) lactic oxidase activity, 50% inhibition occurring at 6.6 x 10(-6)m.