T4 RNA ligase catalyzes the synthesis of ATP β,γ‐bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH2p) (100), clodronate (pCCl2p) (52), and etidronate (pC(OH)(CH3)p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH2–CH2–NH2)p), and alendronate (pC(OH)(CH2–CH2–CH2–NH2)p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E‐AMP in a concentration dependent manner. The K m values and the rate of synthesis (k cat) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26 ± 0.05 mM (0.28 ± 0.05 s−1); clodronate, 0.54 ± 0.14 mM (0.29 ± 0.05 s−1); and etidronate, 4.3 ± 0.5 mM (0.028 ± 0.013 s−1). In the presence of GTP, and ATP or AppCCl2p the relative rate of synthesis of adenosine 5′,5‴‐P1,P4‐tetraphosphoguanosine (Ap4G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH2p) was a very poor substrate for the synthesis of Ap4G. To our knowledge this report describes, for the first time, the synthesis of ATP β,γ‐bisphosphonate analogues by an enzyme different to the classically considered aminoacyl‐tRNA synthetases.