U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is an essential splicing factor with critical roles in recognition of the 3'-splice site. In animals, the U2AF small subunit (U2AF35) can bind to the 3'-AG intron border and promote U2 small nuclear RNP binding to the branch-point sequences of introns through interaction with the U2AF large subunit. Two copies of U2AF35-encoding genes were identified in Arabidopsis (Arabidopsis thaliana; atU2AF35a and atU2AF35b). Both are expressed in all tissues inspected, with atU2AF35a expressed at a higher level than atU2AF35b in most tissues. Differences in the expression patterns of atU2AF35a and atU2AF35b in roots were revealed by a promoter::beta-glucuronidase assay, with atU2AF35b expressed strongly in whole young roots and root tips and atU2AF35a limited to root vascular regions. Altered expression levels of atU2AF35a or atU2AF35b cause pleiotropic phenotypes (including flowering time, leaf morphology, and flower and silique shape). Novel slicing isoforms were generated from FCA pre-mRNA by splicing of noncanonical introns in plants with altered expression levels of atU2AF35. U2AF35 homologs were also identified from maize (Zea mays) and other plants with large-scale expressed sequence tag projects. A C-terminal motif (named SERE) is highly conserved in all seed plant protein homologs, suggesting it may have an important function specific to higher plants.