Clostridium acetobutylicum P262 cells that were growing on lactate and acetate had an NAD-independent lactate dehydrogenase (iLDH) activity of 200 nmol mg protein-1 min-1. Ammonium sulfate precipitation and DEAE cellulose caused a 35-fold purification. Gel filtration indicated that the iLDH had a molecular weight of approximately 55 kDa, but two bands were always observed. Phenyl sepharose could not separate the two proteins, and hydroxyapatite caused a complete loss of activity. The semi-purified iLDH had a V(max) of 13,000 nmol mg protein-1 min-1 and a K(m) value of 3.5 mM for D-lactate. The V(max) and K(m) values for L-lactate were 300 nmol mg protein-1 min-1 and 0.7 mM. The iLDH had a pH optimum of 7.5, was not activated by fructose-1,6-bisphosphate (FDP), and could be coupled to either 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or dichlorophenol-indophenol (DCPIP), but not methyl viologen (MV) or benzyl viologen (BV). The iLDH did not have strong absorbance between 500 and 300 nm, and trichloroacetic acid or acid ammonium sulfate extracts had virtually no fluorescence at 450 nm. The crude extracts also had MTT-linked butyryl- CoA dehydrogenase activity (60 nmol mg protein-1 min-1). The NAD- independent butyryl-CoA dehydrogenase eluted from DEAE-cellulose as two fractions. The yellow fraction was extremely unstable, but the green fraction could be stored for short periods of time at 5°C. The green-colored butyryl- CoA dehydrogenase had strong absorption at 450 nm, and gel filtration indicated that it had a molecular weight of 90 kDa. The NAD-independent butyryl-CoA dehydrogenase could be coupled to MTT, DCPIP, or MV, but not BV. Because the NAD-independent lactate and butyryl-CoA dehydrogenase could both be linked to low potential carriers, these two enzymes may function as oxidation-reduction system in vivo.