Correlation between cell cycle progression and endogenous levels of plant hormones was studied in synchronized tobacco BY-2 cell suspension cultures. Sixteen different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) were extracted using solid-phase anion exchange chromatography in combination with immunoaffinity purification, and quantified by mass spectrometry. No significant correlation could be identified for IAA and ABA. In contrast, there were sharp peaks in the levels of specific cytokinins (zeatin- and dihydrozeatin-type) at the end of the S phase and during mitosis. The levels of other cytokinins analyzed, including zeatins N- and O-glucosides, remained low, suggesting that the increased amounts of their corresponding non-glucosylated form resulted from de novo synthesis. These findings suggest that zeatin- and dihydrozeatin-type cytokinins might play a specific regulatory role in the progression of the plant cell cycle. One hypothesis to explain cytokinin action is based on a specific interaction with kinases that regulate cell cycle progression, as has been recently shown for the cytokinin analogue olomoucine.