Chalcone synthase (CHS) catalyses the first and key regulatory step of flavonoid biosynthetic pathway. A chalcone synthase gene was isolated from Ginkgo biloba leaves using the method of rapid amplification of the cDNA ends (RACE). The full-length cDNA, designated as GbCHS2, is 1,608 bp in length (GenBank accession No. DQ054841) and contains an open reading frame of 1,173 bp encoding a protein of 391 amino acids. Alignment of the predicted amino acid sequence of GbCHS2 has been shown to have high sequence similarity with GbCHS1. All the active sites and active site motifs in GbCHS1 protein were also found in GbCHS2. Correlation analysis between CHS activity and flavonoid accumulation during ginkgo leaf growth indicated that CHS might be the rate-limiting enzyme in the biosynthesis pathway of flavonoids in ginkgo leaves. Results of semi-quantitative RT-PCR analysis showed that flavonoid accumulation paralleled the transcription level of change in chs gene, suggesting chs gene as the specific key gene regulating flavonoid accumulation in ginkgo leaves.