Upon high throughput screening of 6700 microbial fermentation extracts, we discovered a compound, designated orthoformimycin, capable of inhibiting protein synthesis in vitro with high efficiency. The molecule, whose structure was elucidated by chemical, spectrometric, and spectroscopic methods, contains an unusual orthoformate moiety (hence the name) and belongs to a novel class of translation inhibitors. This antibiotic does not affect any function of the 30S ribosomal subunit but binds to the 50S subunit causing inhibition of translation elongation and yielding polypeptide products of reduced length. Analysis by fluorescence stopped flow kinetics revealed that EF-G-dependent mRNA translocation is inhibited by orthoformimycin, whereas, surprisingly, translocation of the aminoacyl-tRNA seems to be unaffected.