Unfractionated mouse long-bone cells prestimulated with rat parathyroid hormone (PTH) for 7 days (PTH-prestimulated long-bone cells; PTH-psLB cells) contained about 20% tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells and about 0.2% TRAP-positive multinucleate cells. When PTH-psLB cells (1.0 x 10(5)/slice) were cultured on dentine slices, about 100 resorption lacunae (pits) per slice were formed by 6 h after the start of culture, while the number of TRAP-positive multinucleate cells on the dentine slices did not increase. When the number of inoculated cells was plotted versus the number of pits formed during the 6-h cultivation, a sigmoid curve was generated. When PTH-psLB cells (1.0 x 10(3)/slice) were cocultured with various mouse stromal cell lines, the MC3T3-G2/PA6 line was found to increase the number of pits. Since this activity was manifested within a short period of time (4 h), and as there was no change in the number of TRAP-positive multinucleate cells, we postulate that the activity of MC3T3-G2/PA6 is exerted on mature osteoclastic cells capable of resorbing bone rather than on some differentiation step of the osteoclast cell lineage. With respect to the mechanism of expression of this activity, it is unlikely that MC3T3-G2/PA6 produces a soluble factor, because no activity was seen when MC3T3-G2/PA6 and PTH-psLB cells were cultured separately across a membrane in a Transwell, These results suggest that cell-to-cell contact and communication between PTH-psLB cells and MC3T3-G2/PA6 may be a crucial event for the stimulation of osteoclastic bone resorption by MC3T3-G2/PA6.