Adenosine kinase is one of the enzymes potentially responsible for the formation of cytokinin nucleotides in plants. Using a zeatin affinity column a 40 kDa protein was isolated from tobacco Bright Yellow 2 (TBY-2) and identified by mass spectrometry as adenosine kinase. The ligand interaction reported here can be disrupted by several other adenine- but not guanine-based purine derivatives. The observed interaction with cytokinins is discussed in view of a putative role for adenosine kinase in TBY-2 cytokinin metabolism. The presented results show for the first time a plant adenosine kinase affinity-purified to homogeneity that was identified by primary structure analysis.