To elicit the roles of cytokinins in the regulation of maturation of Pinus radiata D. Don, the spectrum of endogenous cytokinins and their concentration in the mature buds were analysed using double-solvent extraction, column complex purification and separation, a novel immunoaffinity purification method, normal and reverse phase high-pressure liquid chromatography, enzymatic treatment, radioimmunoassay and electrospray MS/MS spectrometry. We have isolated two novel cytokinin glycosides whose proposed structures are isopentenyladenine-9-(glucopyranosyl riboside), dihydrozeatin-9-(glucopyranosyl riboside) and confirmed the presence of zeatin-9-(glucopyranosyl riboside). We have also found the presence of novel phosphorylated forms of these 3 cytokinin ribosyl-linked glycosides. Quantitative analyses revealed that the cytokinin ribosyl-linked glycosides predominate in P. radiata mature buds. Although cytokinin free base, riboside and nucleotide forms are also present, we could find no evidence of the traditional cytokinin O- or N-glucosides in the conifer buds. Thus, cytokinin metabolism in mature buds of P. radiata is very different from other species previously examined.