A bacterium strain capable of growing on N,N-dimethylformamide (DMF) was isolated using enrichment and isolation techniques. The isolated strain, KUFA-1, was classified as belonging to the genus Alcaligenes which has not been listed as DMF-utilizer previously. Crude soluble extracts of Alcaligenes KUFA-1 grown on DMF were found to contain dimethylformamidase (DMFase). The DMFase was purified to an apparent electrophoretic homogeneity with an overall 38.6-fold purification, a 6.7% yield and a final specific activity of 26.25 mu mol DMF hydrolyzed/min/mg protein. The native DMFase had a relative molecular mass of 180 kDa and was composed of one light-chain (M-r=16,000) and one heavy-chain (M-r=74,000). The optimum pH and temperature were 6.6 and 55 degrees C, respectively. The enzyme was highly specific for DMF. DMFase obeyed Michaelis-Menten kinetics and its K-m and V-max values for DMF were 1.28 mM and 83.3 U/mg, respectively, as determined using a Lineweaver-Burk plot.