A beta-lactamase gene was isolated for the first time from a terrestrial subsurface environment using a combined cultivation and direct cloning strategy. The gene, discovered from 24 m below land surface in Hawaii, was most similar to the penicillinase from Bacillus licheniformis. The resistance gene was confirmed via subcloning and its minimum inhibitory concentration values were measured against several test beta-lactam antibiotics. This study extends the knowledge on resistance to antimicrobials, which may help the efforts to minimize their future threat.