A cDNA clone OsMADS16 was isolated from the rice young inflorescence cDNA expression library by the yeast two-hybrid screening method with OsMADS4 as bait. We have previously shown that the OsMADS4 gene is a member of the PI family and that the MADS-box gene is involved in controlling development of the second and third whorls of rice flowers. The sequence comparison indicated that OsMADS16 belongs to the AP3 family. The OsMADS16 protein contains a PI-derived motif, FAFRVVPSQPNLH, that is a conserved sequence in AP3 family genes at the C-terminal region. In addition, OsMADS16 contains a paleoAP3 motif, YGGNHDLRLG, downstream of the PI-derived motif. The paleoAP3 motif is a consensus sequence in the C-terminal region of the AP3 family genes of lower eudicot and magnolid dicot species. RNA blot analysis showed that the OsMADS16 gene was expressed in the second and third whorls, whereas the OsMADS4 transcripts were present in the second, third, and fourth whorls. These expression patterns of the OsMADS16 and OsMADS4 genes are very similar to those of AP3 and PI, respectively. In the yeast two-hybrid system, OsMADS4 interacted only with OsMADS16 among several rice MADS genes investigated, suggesting that OsMADS4 and OsMADS16 function as a heterodimer in specifying sepal and petal identities. The OsMADS16 protein displayed transcription activation ability in yeast, whereas AP3 did not. It was also shown in yeast that OsMADS16 interacted with PI whereas OsMADS4 did not interact with AP3. These differences between OsMADS16 and AP3 indicate that the functions of the AP3 family genes of monocots and dicots diverged during molecular evolution processes of the B function genes. Deletion analysis showed that the 155-200 amino acid region of the OsMADS16 protein plays an important role in the transcription activation ability.