Two issues were studied: the loss of hormone responsiveness during culture of neonatal rat calvariae under standard culture conditions and the recovery of hormone responsiveness in calvariae previously desensitized with PTH (2.5 U/ml), prostaglandin E2 (PGE2; 1 or 2.5 micrograms/ml), or salmon calcitonin (sCT; 100 mU/ml). Both of these issues were studied in the presence and absence of cortisol (10(-6) M). Culture of neonatal rat calvariae in medium without cortisol resulted in a significant decrease in cAMP responsiveness to PTH, PGE2, and sCT. The addition of cortisol to the culture medium: 1) maintained cAMP responsiveness to PTH at levels approximately 50% of the response of freshly dissected, uncultured calvariae, 2) increased cAMP responsiveness to PGE2 to 120% of the fresh response, and 3) enhanced the loss of cAMP responsiveness to sCT. With regard to the recovery of hormone responsiveness in calvariae previously desensitized with PTH, PGE2, or sCT and then cultured in medium with or without cortisol, the following observations were made. 1) PTH-desensitized calvariae required a 24-h culture period in cortisol to fully recover cAMP responsiveness to PTH. Without cortisol, the recovery was only partial. 2) PGE2-desensitized calvariae were fully recovered after a 24-h culture period in the presence or absence of cortisol. Further experiments indicated that recovery of PGE2-desensitized calvariae was complete after only 2 h of culture in cortisol. 3) sCT-desensitized calvariae did not regain responsiveness to sCT in the presence of cortisol, but partially recovered responsiveness in the absence of cortisol. These results indicate that the recovery of hormone responsiveness of bone in vitro after homologous desensitization with sCT, PTH, or PGE2 follows a distinct pattern for each hormone and suggest that different cellular mechanisms may be involved.