PTH stimulates osteoclastic bone resorption in vivo and in organ culture. We have previously found that if osteoclasts are disaggregated from bone and incubated on bone slices, PTH does not increase bone resorption, but does so if osteoblastic cells are added to the cultures. This suggests that PTH acts primarily on osteoblasts, which are induced by the presence of the hormone to stimulate osteoclastic bone resorption. In the present paper we describe investigations into the mechanism by which osteoblastic cells stimulate osteoclasts. We found that increased resorption could not be accounted for by changes in the bone substrate. Osteoblast-like cells (UMR106) incubated with PTH did, however, release a factor into the culture supernatant that stimulated osteoclastic bone resorption. This factor was stable for at least 7 days when stored at 4 C and survived freeze-thawing, but was inactivated by heating to 65 C for 30 min. Activity was lost entirely after dialysis using a Spectrapor membrane with a mol wt cut-off (MWCO) of 2000. The small size of the molecule was confirmed after ultrafiltration across Amicon filters YM2 and YC05. There was no loss of activity across YM2 (MWCO, 1000), but, in contrast, there was no stimulation in the conditioned medium after ultrafiltration across YC05 (MWCO, 500).