Ultrastructural analyses of the flagellar apparatus suggested that Sphaeroplea, Atractomorpha, the Hydrodictyaceae, and the Neochloridaceae, all of which produce biflagellate motile cells with directly opposed (DO) basal bodies, are allied in an order Sphaeropleales. Recent studies of 18S rDNA sequence data supported an alliance of the DO group, but no data from Sphaeroplea and its allies were included. This investigation presented a test of the phylogenetic hypothesis suggested by the flagellar apparatus evidence using sequence data from the nuclear-encoded small-subunit rDNA (18S) and large subunit rDNA (26S) genes, combined with additional taxon sampling. Results from phylogenetic analyses weakly supported monophyly of biflagellate DO taxa and indicated that pyrenoids with cytoplasmic invaginations are present in numerous distinct lineages. Analysis of both molecular data sets supported a class Chlorophyceae comprised of at least six major groups that generally correspond to currently recognized orders or families: Chaetophorales, Chae- topeltidales, Chlamydomonadales, Sphaeropleales, Sphaeropleaceae, and Oedogoniales. In addition, Cylindrocapsa, Elakatothrix, Treubaria, and Trochiscia formed a seventh chlorophycean clade that is new to science. This investigation demonstrated that the 26S rDNA gene provides more phylogenetic signal, per unit sequence, than the 18S rDNA gene and that combined analysis yields topologies with more robust support than independent analysis of either data set.