We studied the effects of parathyroid hormone (PTH) on PTH parathyroid hormone related peptide (FTHrP) receptor mRNA level, PTHrP binding, and PTH-stimulated cyclic adenosine monophosphate (CAMP) accumulation in osteoblasts, derived from fetal rat calvariae (ROB). Cells isolated during 10-70 minutes of collagenase treatment were seeded at a density of 25,000 cells/cm2 and cultured for 4 days. These cells show a fast increase in CAMP production after stimulation for 5 minutes with 20 nM bovine parathyroid hormone(1-34) (bPTH(1-34)). When ROB are incubated with bPTH(1-34) (0.04-40 nM) for 24 h, a dose-dependent decrease of the P T m H r P receptor mRNA level, PTHrP binding, and PTH-stimulated CAMP accumulation can be observed. Pretreatment of ROB with a high concentration of bPTH(1-34) (40 nM) leads within 15 minutes to a decrease in PTH-stimulated CAMP accumulation. However, it takes 2 3 h before a significant decrease in PTWPTHrP receptor mRNA level can be observed. Also a significant decrease in PTHrP binding is observed after only 4 h of incubation with bPTH(1-34). Compared with bPTH(1-34), pretreatment of ROB with bPTH(3-34) (40 and 100 nM) for 24 h causes smaller decreases in PTH-stimulated CAMP accumulation, PTHrP binding, and in the P T W r P receptor mRNA level. We investigated the possible involvement of the protein kinase A signaling pathway in the regulation of the PTHPTHrP receptor mRNA expression. Both forskolin and (Bu),cAMP decreased FTHrP binding and PTWPTHrP mRNA levels. These observations suggest that chronic activation of the PKA signaling pathway may down-regulate PTWPTHrP receptor expression and thus hormone responsiveness in "normal" osteoblasts. In short, we found that the decrease of the PTH-stimulated CAMP accumulation after long-term pretreatment with bPTH(1-34) is correlated with both PTHPTHrP receptor mRNA level and PTHrP binding. These data also suggest that the initial desensitization ( ~3 0 minutes) of PTH-stimulated CAMP responsiveness by pretreatment with a high concentration of bPTH(1-34) (40 nM) is not dependent on the number of available PTHPTHrP receptors. The protein kinase A signaling pathway is involved in the regulation of the PTHPTHrP receptor, but, regarding the effect of bFTH(3-34), other signaling systems are also involved. (J Bone Miner Res 19%;