Ricin (RIC), modeccin (MOD), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are protein toxins that enter cells by receptor-mediated endocytosis. After intracellular transport and membrane translocation to the cytosol, these toxins inhibit protein synthesis by enzymatically removing a specific adenine residue from ribosomal RNA (RIC, MOD), or by ADP-ribosylation of elongation factor-2 (PE, DT). Recently, Thompson and Pace (1992) reported that AZT (3'-azido-3'-deoxythymidine) inhibited RIC toxicity in Vero cells, and this inhibition was not due to a block of RIC enzymatic activity. This paper extends these findings and examines the effects of AZT treatment on the toxicities of other protein toxins in Chinese hamster ovary (CHO) and Vero cell lines. AZT treatment did not significantly alter the toxicity of DT or MOD in either cell line, but it markedly reduced RIC and PE toxicity in both cell lines. The ID50 values (concentration of toxin required to inhibit protein synthesis by 50%) for RIC and PE in CHO cells increased approximately 6.5- and 12.5-fold, respectively; while in Vero cells the ID50 values increased ca. 8.5- and 4.5-fold, respectively. Results of further studies revealed differences in the mechanisms by which AZT inhibited RIC and PE toxicity. Results of cell-free translation indicated that, unlike its effects on RIC, AZT blocked the ability of PE to perform its enzymatic activity. As AZT did not block RIC enzymatic activity, we examined the effects of AZT on earlier steps in the RIC intoxication process. AZT treatment did not inhibit cell-surface binding or internalization of [125I]-RIC. Results of kinetic studies showed that when AZT was incubated with cells at the time of RIC exposure, it caused no major change in the lag phase, during which RIC reaches the site of translocation. However, it clearly reduced the subsequent first-order reduction in the rate of protein synthesis, suggesting an effect on translocation. Monensin (an ionophore that perturbs intracellular trafficking and increases the toxicities of RIC and PE) reduced AZT protection against both toxins. Nocodazole and colchicine (agents that disrupt microtubules and some routes of intracellular trafficking) reduced the ability of AZT to inhibit RIC, but not PE, toxicity. In summary, our results suggest that (1) AZT acts within the cytosol to inhibit (directly or indirectly) the enzymatic action of PE, and (2) the AZT inhibition of RIC cytotoxicity does not involve perturbations of RIC cell-surface binding, internalization, or enzymatic activity but might result from an alteration in RIC translocation.