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Maize ribosome-inactivating protein 1 (RIP1) is an abundant 32-kDa protein that can depurinate a specific adenine residue on large 28S ribosomal RNA. This irreversible modification of the ribosomal RNA inhibits translation. We have tested both the inactive precursor, proRIP1, and active RIP1 for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well characterized non-pathogenic species, were treated with RIP1. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed that there was only one growing hyphal tip per conidium. In a developmental time-course both fungal species were affected by RIP1 at the transition from pre- to post-divisional growth. We made two site-directed mutants of RIP1 and used them to determine if the ribosome-inactivating activity of RIP1 plays a role in its antifungal activity. The mutants had reduced ribosome-inactivating activity in translational inhibition assays in vitro and showed no antifungal activity against A. nidulans. When A. nidulans conidia were treated with active and mutant RIP1 depurination was observed in the ribosomal RNA isolated from germlings. RIP1 is larger than compounds known to be taken up by fungal cells, so we examined whether RIP1 varients were taken up by A. nidulans cells by tagging the proteins with the fluorophore Texas Red. The germlings accumulated equivalent amounts of active and mutant RIP1 inside the cell and at the cell periphery. An A. nidulans genetic mutant, fadAG203R, was found to be resistant to maize RIP1 antifungal activity and had two-fold less RIP1 localization than normal germlings. To test whether amount of proRIP1 in the maize kernel was correlated with resistance to A. flavus, kernels with high and low levels of proRIP1 were inoculated in the field with an A. flavus strain expressing GFP. We observed a positive correlation between the amount of proRIP1 in the kernel and resistance to fungal infection. This work supports the hypothesis that maize RIP1 has antifungal activity against Aspergillus species.