Adenosine kinase (ADK), a key enzyme in the regulation of the intracellular level of adenosine is also speculated to be responsible for the conversion of cytokinin ribosides to their respective nucleotides. To elucidate the role of ADK in the cytokinin metabolism of tobacco BY-2 cells (Nicotiana tabacum cv. "Bright Yellow-2"; TBY-2), we have identified and characterized the full-length cDNAs encoding four ADK isoforms of N. tabacum and determined their catalytic properties. The four TBY-2 ADK isoforms (designated 1S, 2S, 1T, and 2T) display a high affinity for both adenosine (Km 1.88-7.30 microm) and three distinct types of cytokinin ribosides: isopentenyladenosine; zeatin riboside; and dihydrozeatin riboside (Km 0.30-8.71 microm). The Vmax/Km values suggest that ADK2S exhibits in vitro an overall higher efficiency in the metabolism of cytokinin ribosides than the other three isoforms. The expression pattern of NtADK genes is modulated significantly during the cell cycle. We suggest that the increased transcript accumulation of NtADK coupled to an increased ADK activity just prior to mitosis is associated with a very active cytokinin metabolism at that phase of the cell cycle of synchronized TBY-2 cells.