The modification of proteins by reducing sugars through the process of non-enzymatic glycation is one of the principal mechanisms by which hyperglycaemia may precipitate the development of diabetic complications. Fn3K (fructosamine 3-kinase) and Fn3KRP (Fn3K-related protein) are two recently discovered enzymes that may play roles in metabolizing early glycation products. However, although the activity of these enzymes towards various glycated substrates has been established, very little is known about their structure-function relationships or their respective mechanisms of action. Furthermore, their only structural similarities noted to date with members of other kinase families has been with the bacterial aminoglycoside kinases. In the present study, we employed affinity labelling with the ATP analogue FSBA {5'-p-[(fluorosulfonyl)benzoyl]adenosine} to probe the active-site topology of Fn3KRP as an example of this enigmatic family of kinases. FSBA was found to modify Fn3KRP at five distinct sites; four of these were predicted to be localized in close proximity to its ATP-binding site, based on alignments with the aminoglycoside kinase APH(3')-IIIa, and examination of its published tertiary structure. The results of the present studies provide evidence that Fn3KRP possesses an ATP-binding domain that is structurally related to that of both the aminoglycoside kinases and eukaryotic protein kinases.