Flavonoid 3¡¦,5¡¦-hydroxylase (F3¡¦,5¡¦H) is the key enzyme that catalyzesthe anthocyanin biosynthesis pathway for the expression of blue or purple flower color. The garden crops of Verbena x hybrida were used to clone its F3¡¦,5¡¦H gene for the investigation of flower color engineering and regulatory program. Degenerate primers were designed from the conservative regions of other published F3¡¦,5¡¦H genes to amplify the expectant DNA fragment . A full-length cDNA of the F3¡¦,5¡¦H gene designated VhFH1 (AY604727) was cloned by the method of 5¡¦and 3¡¦ RACE, and its genomic DNA sequence was isolated by the IPCR strategy. Nucleotide sequence alignment revealed that VhFH1 contains two introns and a 1542 bp open reading frame encoding a polypeptide of 514 amino acid residues, and there could be a promoter sequence with TATA box signal in the upstream of the transcription start site. The amino acid sequence of VhFH1 was compared with the previous reported F3¡¦,5¡¦H and showed between 50¢Mand 77¢Midentity with those species. The expectant molecular mass and isoelectric point of VhFH1 protein is 57 KD and 7.69, respectively. There are three typical motifs of the F3¡¦,5¡¦H that belongs to the cytochrome P450 proteins in the VhFH1 predicted protein. According to the above-mentioned conjecture, VhFH1 is a full-length cDNA of the F3¡¦,5¡¦H gene in the V. hybrida. This cDNA fragment was inserted into the plant expression vector pCambia 1304 and could be detected the expression in E. coli by RT-PCR and protein electrophoresis. It is practicable to transform the horticultural plants with these vectors to create novel flower colors in the future. Furthermore, transcripts of the F3¡¦,5¡¦H gene were detected in the blue, purple and white flowers but not in the red one as revealed by RT-PCR. These results are advantageous in the further investigation of regulatory factors of the anthocyanin biosynthesis pathway in the V. hybrida.