The aim of this study was to evaluate the different bone resorption markers, total pyridinoline (Pyr) and total deoxypyridinoline (Dpyr), assessed by a HPLC method, free Dpyr, assessed by a new immunoassay, and urinary excretion of hydroxyproline (OH-proline), in postmenopausal osteoporotic women during long-term treatment with pamidronate. A total of 60 postmenopausal women with previous distal forearm fracture were included in this 12-month placebo-controlled and double-blind study, where intermittent oral pamidronate, 75 or 150 mg, or placebo were given daily for 4 weeks, every 16 weeks. After 1 week a significant reduction in urinary excretion of total Dpyr was observed in the group treated with 150 mg pamidronate compared to the placebo (p < 0.01) and to the 75-mg group (p < 0.001). A maximal 50.4% decrease in total Dpyr, (p < 0.0001 compared to the placebo group, p < 0.01 compared to the 75-mg group), was observed after 3 weeks of treatment with 150 mg pamidronate, and this decrease persisted to week 52. After 4 weeks of treatment with 150 mg pamidronate the maximal decrease in free Dpyr was only 26.5%, which persisted during 12 months of treatment. Decreases in urinary excretion of total Pyr and OH-proline were less than the decreases in total Dpyr. The correlation between total Dpyr (HPLC method) and free Dpyr (Pyrilinks-D assay) at baseline was r = 0.91. Total Dpyr assessed by the HPLC method reflects the pamidronate-induced decrease in bone resorption, and the changes in this resorption marker were more pronounced than changes in free Dpyr, total Pyr and OH-proline. In this study free Dpyr analysis was less suitable for reflecting bone resorption during bisphosphonate therapy.