A novel osteoclast generation and bone resorption assay system is described in which enhanced osteoclastic generation and bone resorption is induced in porcine bone marrow cell cultures cultured in low-serum medium supplemented with fibroblastic cell (L929) conditioned medium (CM). Numerous osteoclasts, which could be identified by TRAP staining and specific labelling with 121F antibody, were generated in a time-dependent and L929-CM concentration-dependent fashion. A specific antibody against murine M-CSF/CSF-1 abolished osteoclast formation indicating that M-CSF is the essential component of the L929-CM driven osteoclast generation. Culturing on devitalized bone slices resulted in extensive osteoclast-mediated resorption as visualized microscopically. After 16 days in culture, practically the entire bone slice surface was excavated by the osteoclastic cells. Bone resorption could be monitored with time using a novel enzyme-linked immunoassay measuring type I collagen N-telopeptides in culture supernatants. Release of collagen fragments from the slices was paralleled by osteoclastic secretion of TRAP. Salmon calcitonin significantly inhibited collagen fragment and TRAP release. 1,25-Dihydroxyvitamin D3 greatly promoted osteoclast generation and subsequent bone resorption, but its presence was not essential for this process to occur.