Production of ?-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to ?-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D ?-lactamases capable of hydrolyzing a wide array of ?-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinct structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D ?-lactamases. In conclusion, these enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.