Aminoglycoside 3'-phosphotransferases [APH(3')s] phosphorylate aminoglycoside antibiotics, a reaction that inactivates the antibiotics. These enzymes are the primary cause of resistance to aminoglycosides in bacteria. APH(3')-Ia operates by a random-equilibrium BiBi mechanism, whereas APH(3')-IIIa catalyzes its reaction by the Theorell-Chance mechanism, a form of ordered BiBi mechanism. Hence, both substrates have to be present in the active site prior to the transfer of phosphate by both mechanisms. Four bisubstrate analogues, compounds 1-4, were designed and synthesized as inhibitors for APH(3')s. These compounds are made of adenosine linked covalently to the 3'-hydroxyl of neamine (an aminoglycoside) via all-methylene tethers of 5-8 carbons. The K(i) values measured for these compounds indicated that affinities of APH(3')-Ia and APH(3')-IIa for compounds 2 and 3 (six- and seven-carbon tethers, respectively) were the best, and the inhibition constants for the two were comparable.