4-(Cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (CMK or YchB), the fourth enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway, phosphorylates the 2-hydroxyl group of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol in the presence of ATP. Two isogenes encoding CMK (GbCMK1 and GbCMK2) were cloned and characterized from Ginkgo biloba. The activities of both isozymes were confirmed by complementation assay using Escherichia coli NMW29, a ychB knock-out mutant. The transcript profiles of GbCMKs in the radicles and the cotyledons of the cultured Ginkgo biloba embryos demonstrated that the transcript levels of GbCMK1 were similar in both organs, whereas that of GbCMK2 was predominantly high in the ginkgolide-synthesizing radicles. Selective increases in the transcript abundance of GbCMK2 in the radicles, induced by light and methyl jasmonate treatments, were observed. These differential induction patterns of the transcripts imply GbCMK1 and GbCMK2 respectively have high correlations with the primary and the secondary metabolisms. The transit peptides of both isozymes delivered the fused green fluorescent protein (GFP) into the chloroplast in the Arabidopsis and the Nicotiana transient expression systems; interestingly, the transit peptide of GbCMK1 delivered the GFP protein into the cytosol and the nucleus in addition to the chloroplasts.