Abstract: The main aim of the present study is to further characterize a new dehalogenase enzyme found in the crude extracts from Rhodococcus sp. The ability of the enzyme to catalyze the dehalogenation of various halogen-substituted organic acids was investigated and the highest activity was found with 3-chloropropionic acid as a sole carbon source in the growth medium. The enzyme followed Michaelis-Menten kinetics and the Km for 3-chloropropionic acid was 0.2 mM. Maximum activity was found at pH 7.6 at 30°C. The enzyme activity in the cell-free extract was unaffected by diaminoethane tetraacetic acid (EDTA), dithiothreitol (DTT) or by Mn and Zn ions but was reduced by HgCl2 (70%) and Pb(NO3)2 (80%). The enzyme removed the chlorine atom present on a number of 3- and 4-carbon alkanoic acids if the halogen was on the β-position.