A systematic method has been developed to assess bone resorption, matrix formation, and calcification in a single calvarial culture from 20-day-old chick embryos. The bones were prelabeled with 45Ca by an injection into the egg white before culture. In the last 2 h of incubation, the bones were further labeled with both 3H-proline and 3H-tetracycline. Bone resorption was assessed as 45Ca release into the medium. Collagen synthesis was measured by the incorporation of 3H-proline into collagenase-digestible protein (CDP). Since tetracycline has been commonly used as an in vivo marker for osteoid calcification, we assessed in vitro calcification as the uptake of 3H-tetracycline into bone. By using this method, we studied the effects of prostaglandin E2 (PGE2) and indomethacin, which inhibits biosynthesis of PGE2, on bone resorption and formation. The cultured bone synthesized approximately 300 ng of PGE2 during every 24 h of incubation, and indomethacin (10(-6) M) completely inhibited the synthesis. In indomethacin-treated cultures, % 45Ca release and the labeling of CDP were significantly reduced. These reductions were not seen when exogenous PGE2 (10(-9) M) comparable to its endogenous level was added along with the indomethacin. Furthermore, 10(-8) to 10(-5) M PGE2 increased % 45Ca release and the CDP labeling. In addition, the uptake of 3H-tetracycline into the cultured bone was also enhanced by PGE2. In devitalized calvaria, PGE2 had no effect on 3H-tetracycline uptake, suggesting that the stimulative effect on PGE2 was cell-mediated.(ABSTRACT TRUNCATED AT 250 WORDS)