Part of the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) gene (rbcL) was sequenced from three fern species: Adiantum capillus-veneris, Botrypus strictus, and Osmunda cinnamomea var. fokiensis. This region included 1,333 base pairs, about 90% of the gene. Maximum likelihood analysis of the deduced amino acid sequences indicated that (1) Botrypus (Ophioglossaceae) clustered monophyletically with other ferns (Adiantum, Angiopteris, Osmunda); the closest relative to Botrypus among the three species was Osmunda, which did not support the hypothesis that the Ophioglossaceae are linked to the progymnosperm-seed plant lineage. (2) Eusporangiate ferns containing Botrypus (Ophioglossaceae) and Angiopteris (Marattiaceae) were a paraphyletic group. (3) Seed plants and the four fern species examined formed a monophyletic group, but ferns and bryophytes (liverwort) did not. Variations in rates of substitution for synonymous and nonsynonymous codons were found in fern lineages.