Zoledronic acid is a new, highly potent bisphosphonate drug under clinical evaluation. A radioimmunoassay has been developed to determine zoledronic acid concentration in human serum, plasma, and urine. The assay utilizes rabbit polyclonal antisera against a zoledronic acid-BSA conjugate and a [125I]zoledronic acid derivative as tracer in a competitive format adapted to microtiter plates. The assay shows a LLOQ 0.4 ng/ml in serum or plasma (interassay%CV=17%, accuracy 97%), 5 ng/ml in urine (21%, 98%). In 23 patients receiving 4, 8 or 16 mg of zoledronic acid, drug concentrations in plasma were dose proportional and showed a multiphasic profile, followed by a prolonged gradual decline to concentrations near the LLOQ. Zoledronic acid disposition in plasma and the recovery of only 40-50% of the dose in urine are consistent with the rapid and extensive uptake by and slow release from bone in parallel with renal clearance, typically shown by bisphosphonates.