In the present study we compared the first generation non-nitrogen-containing bisphosphonate, clodronate with second and third generation nitrogen-containing bisphosphonates, pamidronate and zoledronic acid in dynamic rat osteoclast resorption and apoptosis assays and in human mesenchymal stem cell-derived osteoblast assay. We found that due to high bisphosphonate-bone binding affinity, bone surface exposure to clodronate for 3 min. had maximal resorption inhibition. The mechanism of action of both clodronate and zoledronic acid involved osteoclast apoptosis, whereas pamidronate had only minor apoptotic effect at dosages, which readily inhibited resorption. Zoledronic acid was not metabolised into an intracellular ATP-analogue in vitro in contrast to clodronate. All bisphosphonates had a dose-dependent inhibitory effect on the human bone marrow mesenchymal stem cell (hMSC)-derived osteoblast calcium deposition. None of the compounds had inhibitory effect on hMSC differentiation. Zoledronic acid was the most potent of all three bisphosphonates in terms of both apoptosis induction and resorption inhibition. Zoledronic acid efficacy might thus use its capacity to trigger osteoclast apoptosis in an unknown, but similar manner to that of the non-nitrogen-containing bisphosphonates. It appears that zoledronic acid has properties of both bisphosphonate classes and could well be the first member of a new class of bisphosphonates, by definition.