Several omega-laurolactam degrading microorganisms were isolated from soil samples. These strains were capable of growing in a medium containing omega-laurolactam as sole source of carbon and nitrogen. Among them, five strains (T7, T31, U124, U224, and U238) were identified as Cupriavidus sp. T7, Acidovorax sp. T31, Cupriavidus sp. U124, Rhodococcus sp. U224, and Sphingomonas sp. U238, respectively. The omega-laurolactam hydrolyzing enzyme from Rhodococcus sp. U224 was purified to homogeneity, and its enzymatic properties were characterized. The enzyme acts on omega-octalactam and omega-laurolactam, but other lactam compounds, amides and amino acid amides, cannot be substrates. The enzyme gene was cloned, and the deduced amino acid sequence showed high homology with 6-aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12) from Arthrobacter sp. KI72 and Pseudomonas sp. NK87. Enzymatic synthesis of 12-aminolauric acid was performed using partially purified omega-laurolactam hydrolase from Rhodococcus sp. U224.