Rapid alkalinization factors (RALFs) are recently reported active peptide hormones and are considered to play important roles in plant development. We previously identified a differentially expressed cDNA fragment between cabbage flower buds of sterility lines and its maintainer line, which showed significant homology to Arabidopsis RALFL9. The novel RALF cDNA (BoRALF1) was isolated from broccoli flower buds by EST assembly. The open reading frame (ORF) comprises 240 bp, encoding a small putative preprotein of 79 amino acids (molecular weight of 8.72 kDa and a pI of 7.8), which contains the mature polypeptide at its C terminus. BoRALF1 shares 70.3% identity with Arabidopsis RALFL9, but has only moderate similarity with functionally characterized RALFs (ranging from 16.2% to 38.0%). BoRALF1 shows typical features of RALFs, including the 28-aa signal peptide, typical arrangement of four position conserved cysteines, the YIXY motif and a similar secondary structure. RT-PCR studies of different tissues and promoter-GUS fusions confirmed that BoRALF1 is expressed strictly in mature pollen grains and in the anther cells around the loculi. Based on in vivo transient assays, we found that BoRALF1 appears to be largely localized in the plasma membrane. Although the function of BoRALF1 remains to be determined, our experiments confirm the presence of RALF peptide in broccoli, and suggest it could have a role in anther or pollen development.