<jats:p> Many known antibiotics target the translational apparatus, but none of them can selectively inhibit initiation of protein synthesis and/or is prokaryotic-specific. This article describes the properties of GE81112, an effective and prokaryotic-specific initiation inhibitor. GE81112 is a natural tetrapeptide produced by a <jats:italic>Streptomyces</jats:italic> sp. identified by an <jats:italic>in vitro</jats:italic> high-throughput screening test developed to find inhibitors of the prokaryotic translational apparatus preferentially acting on steps other than elongation. <jats:italic>In vivo</jats:italic> GE81112 inhibits protein synthesis but not other cell functions such as DNA duplication, transcription, and cell wall synthesis. <jats:italic>In vitro</jats:italic> GE81112 was found to target the 30S ribosomal subunit and to interfere with both coded and noncoded P-site binding of fMet-tRNA, thereby selectively inhibiting formation of the 30S initiation complex.