We have investigated signaling (cAMP) and anabolic responses (mineralization of extracellular matrix [ECM]) to parathyroid hormone (PTH) in long-term (30 days) cultures of MC3T3-E1 cells, a murine model of osteoblast differentiation. Expression of PTH/PTH-related peptide receptor (PTH1R) mRNA is detected early and remains relatively constant for 2 weeks with somewhat higher levels observed during the second half of the culture period. In contrast to the relatively stable PTH1R mRNA expression, the cAMP response to PTH varies markedly with no response at day 5 and a marked response (80-fold versus control) by day 10. Responsiveness to PTH remains elevated with fluctuations of 30- to 80-fold stimulation throughout the remainder of the culture period. The timing and duration of PTH treatment to achieve in vitro mineralization of ECM was evaluated. When continuous PTH treatment was initiated before day 20, mineralization decreased. If continuous PTH treatment began on or after day 20, mineralization was unaffected. However, if treatment began on day 20 and then stopped on day 25, mineralization on day 30 was increased 5-fold. This mineralization response to intermittent PTH was confirmed in primary cultures of murine and human osteoblastic cells. These data provide a potential basis for understanding the differential responses to PTH (anabolic versus catabolic) and indicate the developmental temporal variance of anabolic and catabolic responses. Since cAMP signaling was relatively unchanged during this interval (day 10- 30) and stimulation of adenylate cyclase only partially mimicked the PTH effect on increased mineralization, other signaling pathways are likely to be involved in order to determine the specific anabolic response to short-term PTH treatment during the differentiation process.