The mechanism of the novel enzymatic dehalogenating action of (R)-3-chloro-1,2-propanediol [monochlorohydrin (MCH)] was investigated. The (R)-MCH-dechlorinating enzyme system of Alcaligenes sp. DS-S-7G, which stereoselectively assimilated (R)MCH from the racemate, was composed of two components (Enzyme 1 and Enzyme 2). Enzyme 1 was a flavoprotein with a relative molecular mass (M(r)) of 70,000 and was composed of two kinds of polypeptides (58,000 and 16,000). The enzyme exhibited activity for converting (R)-MCH to hydroxyacetone with the liberation of chloride ions under aerobic conditions. On the other hand, Enzyme 2 with an M(r) of 86,000, which was also composed of two kinds of polypeptides (33,000 and 53,000), showed no dechlorinating activity for (R)MCH. However, in the presence of NAD(+), when Enzyme 1 was conjugated with Enzyme 2 in the (R)MCH-dechlorinating reaction, the co-operative dechlorinating activity was four to five times higher than that by Enzyme 1 alone. (R)-MCH was finally degraded to acetic acid and formic acid by the joint action of the two enzymes. These facts indicate that (R)-MCH is oxidatively dechlorinated by the two enzymes in the presence of NAD(+) in Alcaligenes sp. DS-S-7G.