The structure-activity relationship for Chinese hamster adenosine kinase (AK) was examined by making systematic deletions from the N- and C-terminal ends. The first 16 a.a. residues from the N-terminal end, which likely form a random coil, can be deleted without any effect on AK activity or stability. The successive removal of the next 11 residues, which stabilize the first beta structure of the protein, leads to a progressive loss of AK activity from 100 to about 3%. The loss in activity is accompanied by increasing thermal instability and a slight increase in the K(m) for adenosine. All deletions beyond residue M28, which should cause disruption of the tertiary structure, are devoid of AK activity. The residues at the C-terminal end form a substructure involved in the stability of the "adenosine 2 binding site" and removal of any residues results in significant loss of activity. Successive removal of the first 10 residues from this end causes progressive decrease in AK activity to about the 2% level, accompanied by a five-fold increase in the K(m) for ATP, supporting the view that the adenosine 2 binding site located near the C-terminal end is the ATP binding site. All deletions beyond residue R348, which forms two salt bridges with the ATP binding site, are inactive. Site-directed replacement of an aspartic acid residue (D316), which is postulated to function in the transfer of phosphate from ATP to adenosine by either asparagine or glutamic acid, leads to complete loss of activity, supporting the proposed role of D316 as the catalytic base.