Metolachlor (2-chloro-6′-ethyl-N-(2-methoxy-1-methylethyl)aceto-o- toluidide) is a pre-emergent chloroacetanilide herbicide used to control broadleaf and annual grassy weeds in a variety of crops. The S enantiomer, S-metolachlor, is the most effective form for weed control. Although the degradation of metolachlor in soils is thought to occur primarily by microbial activity, little is known about the microorganisms that carry out this process and the mechanisms by which this occurs. This study examined a silty-clay soil (a Luvisol) from Spain, with 10 and 2 year histories of metolachlor and S-metolachlor applications, respectively, for microorganisms that had the ability to degrade this herbicide. Tis paper reports the isolation and characterization of pure cultures of Candida xestobii and Bacillus simplex that have the ability to use metolachlor as a sole source of carbon for growth. Species assignment was confirmed by morphological and biochemical criteria and by sequence analysis of 18S and 16S rRNA, respectively. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses indicated that C. xestobii degraded 60% of the added metolachlor after 4 days of growth and converted up to 25% of the compound into CO2 after 10 days. In contrast, B. simplex biodegraded 30% of metolachlor following 5 days of growth in minimal medium. In contrast, moreover, the yeast degraded other acetanilide compounds and 80% of acetochlor (2-chloro-N-ethoxymethyl- 6′-ethylaceto-o-toluidide) and alachlor (2-chloro-2′,6′- diethyl-N-methoxymethylacetanilide) were degraded after 15 and 41 h of growth, respectively. The results of these studies indicate that microorganisms comprising two main branches of the tree of life have acquired the ability to degrade the same novel chlorinated herbicide that has been recently added to the biosphere.