Prolonged use of calcitonin (CT) in vivo leads to a loss of biological response (escape). To understand the molecular basis of this phenomenon, we examined desensitization of calcitonin receptors and down-regulation of adenylate cyclase response to CT in T47CD cells after a preincubation with CT. Preincubation with salmon or human CT (sCT, hCT) for 3 h led to a concentration-dependent loss of [125I]-sCT binding and a similar loss of adenylate cyclase response to a maximal stimulatory dose of sCT. At the same time there was an increased basal activity of the adenylate cyclase. After 24 h preincubation with sCT, basal cAMP levels fell considerably but not to basal levels. Time course experiments showed a delayed decline of maximally sCT-stimulated cAMP levels, which started after 1 h, while binding values declined over the first 60 min to one-third of the original values. Upon removal of CT from the medium, recovery of hormone binding occurred in parallel with the recovery of the adenylate cyclase response to sCT. T47D cells incubated with 25 μmol/l monensin, a lysosomal inhibitor, showed a persistent [125I]-sCT binding after removal of hormone, consistent with diminished intracellular receptor degradation. However, despite persistent binding to the cells, basal and stimulated cAMP levels dropped in the same manner as seen in controls. Our experiments support the view that tight binding of CT to its receptor stimulates adenylate cyclase in T47D cells, until receptors are removed by internalization. The physiological role of internalization of the CT receptor might be to end continuous stimulation of the adenylate cyclase, which occurs after binding.