Pepocin, isolated from Cucurbita pepo, is a ribosome-inactivating protein (RIP). RIPs site-specifically recognize and depurinate an adenosine at position 4324 in rat 28 S rRNA, rendering the ribosome incapable of interacting with essential elongation factors. Aptamers that target pepocin were isolated from a degenerate RNA pool by in vitro selection. A conserved hairpin motif, quite different from the sequence of the toxin-substrate domain in rat 28 S rRNA, was identified in the aptamer sequences. The aptamers selectively bind to pepocin with dissociation constants between 20 and 30 nM and inhibit the N-glycosidase activity of pepocin on rat liver 28 S rRNA. Competitive binding experiments using aptamer variants suggest that the conserved hairpin region in the anti-pepocin aptamer binds near the catalytic site on pepocin and prevents the interaction of pepocin and 28 S rRNA. Anti-RIP aptamers have potential use in diagnostic systems for the detection of pepocin or could be used as therapy to prevent the action of pepocin in mammalian cells.