The nucleotide sequence of the small-subunit rRNA (18S rRNA) coding gene in the higher fungus Protomyces inouyei contains two group I introns. This is the first report of two group I introns in the 18S rRNA coding region. Based on the comparison of the two introns of Protomyces inouyei with those of the green alga Ankistrodesmus stipitatus, and the other two higher fungi Pneumocystis carinii and Ustilago maydis, the Protomyces introns are group I introns containing the highly conserved sequence elements P, Q, R, and S. Intron A of Protomyces inouyei is located in the same position as in Pneumocystis carinii while intron B shares the location with that in Ustilago maydis. The phylogenetic relationships strongly support horizontal transfer of these group I introns.