The enzymatic mechanism of a small ribosome-inactivating protein, gamma-momorcharin, purified from the seeds of Momordica charantia, has been characterized. By SDS-polyacrylamide and electrospray ionization mass spectrometry, its molecular weight was measured to be 11,500 daltons which is much lower than other RIPs known to date. It can inhibit the protein synthesis in the rabbit reticulocyte cell-free system with ID50 of 55 nM. When rat liver ribosome was incubated with gamma-momorcharin, a diagnostic RNA fragment appeared on the gel after rRNAs were treated with acid aniline. Sequencing of the RNA fragment indicates that the action site of gamma-momorcharin in 28S ribosomal RNA of rat liver is at a specific adenosine (position 4324), which is in a highly conserved loop of 28S rRNA.