Biotinyl-CoA synthetase, the first enzyme involved in biotin degradation, was purified from a cell-free extract of a biotin-degrading bacterium, Mycoplana sp. No. 166. The purification procedures comprised polyethyleneimine treatment, ammonium sulfate fractionation, and DEAE-Sepharose, Blue-Sepharose, Sephadex G-100, and FPLC (Mono Q HR 5/5) column chromatographies. The enzyme was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a monomeric enzyme with a molecular weight and an isoelectric point of about 55,000 and 4.85, respectively. The purified enzyme catalyzed the stoichiometric conversion of biotin, ATP, and CoA into biotinyl-CoA, AMP, and PPi. Dethiobiotin and actithiazic acid, a synthetic biotin analog, were also effective as substrates. Other properties of the purified enzyme were also investigated.