In plants, 14-3-3 proteins are key regulators of primary metabolism and membrane transport. Although the current dogma states that 14-3-3 isoforms are not very specific with regard to target proteins, recent data suggest that the specificity may be high. Therefore, identification and characterization of all 14-3-3 (GF14) isoforms in the model plant Arabidopsis are important. Using the information now available from The Arabidopsis Information Resource, we found three new GF14 genes. The potential expression of these three genes, and of two additional novel GF14 genes (Rosenquist et al., 2000), in leaves, roots, and flowers was examined using reverse transcriptase-polymerase chain reaction and cDNA library polymerase chain reaction screening. Under normal growth conditions, two of these genes were found to be transcribed. These genes were named grf11and grf12, and the corresponding new 14-3-3 isoforms were named GF14omicron and GF14iota, respectively. The gene coding for GF14omicron was expressed in leaves, roots, and flowers, whereas the gene coding for GF14iota was only expressed in flowers. Gene structures and relationships between all members of the GF14 gene family were deduced from data available through The Arabidopsis Information Resource. The data clearly support the theory that two 14-3-3 genes were present when eudicotyledons diverged from monocotyledons. In total, there are 15 14-3-3 genes (grfs 1–15) in Arabidopsis, of which 12 (grfs 1–12) now have been shown to be expressed.