Fungal pathogens are increasing in prevalence due to an increase in resistant strains and the number of immunocompromised humans. Candida albicans is one of these pathogens, and approximately 40% of strains contain a group I self-splicing intron, which is a potential RNA drug target, in their large subunit rRNA precursor. Here, we report that Hoechst 33258 and derivatives thereof are selective inhibitors of C. albicans group I intron self-splicing with an IC50 of 17 microM in 2 mM Mg2+. Chemical probing of the intron in the presence of Hoechst 33258 reveals that the folding of several nucleotides in the P4/P6 region of the intron is affected. A nucleotide near the J4/5 region is protected from chemical modification in the presence of Hoechst 33258 and several nearby are more reactive; this suggests that this region is the molecule's binding site. These results expand the available information on small-molecule targeting of RNA and suggest that the RNA-targeting scaffold provided by Hoechst may prove valuable in designing compounds that inhibit the functions of RNA.