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Didemnin B is an inhibitor of eukaryotic protein synthesis and displays potent anti-viral, antineoplastic and immunosuppressive properties. We have established a structure-activity relationship for the inhibition of protein synthesis by didemnin B in vitro. The SAR data obtained indicate that the native conformation of didemnin B is critically important for activity. The relative order of potency of didemnin analogs for protein synthesis inhibition in vitro correlates well with that in intact cells; thus, the inferences drawn regarding SAR for translation inhibition in vitro should also hold true in cells. Previous mechanistic studies had shown that didenmin B specifically inhibits translation elongation by preventing the eEF-2-mediated translocation step and that this inhibitory activity is mediated by eEF-1alpha in vitro . We have examined this mechanism in greater detail. We have shown that didemnin B binds to the ribosome complex, with modest affinity (Kd ∼ 4 muM), in an eEF-1alpha dependent manner. In the presence of didemnin B, eEF-2 is blocked from binding to the ribosome complex and therefore translocation is inhibited. We propose that the interaction between didemnin B and eEF-1alpha on the ribosome locks the ribosome complex in a conformation unfavorable for eEF-2 binding; alternatively, on associating with ribosome-bound eEF-1alpha, didemnin B occupies eEF-2's binding site, thus physically occluding eEF-2 from accessing the ribosome. Currently, there is no way to distinguish between these modes of inhibition. We have shown that inhibition of cellular protein biosynthesis by didemnin B is sufficient to inhibit cell proliferation, but not sufficient to trigger apoptosis. Thus, at low concentrations, didemnin B is a cytostatic agent, but not cytotoxic.