The hormone-sensitive adenylate cyclase system of a cloned bone cell line (UMR-106) derived from a rat osteosarcoma was compared in preparations from cells of early passages (less than 50) and cells maintained in continuous culture for over two years (late passages). Late passage cells showed greater calcitonin (CT)-stimulated adenylate cyclase activity than did early passages, whereas stimulation by PTH and the beta-adrenergic agonist isoproterenol decreased in late passages. Hormone concentrations giving half-maximal stimulation were the same in early and late passages. Stimulation by agents (GTP and fluoride) which act at the stimulatory guanine nucleotide regulatory component (Ns) of adenylate cyclase was equivalent in early and late passages. Forskolin stimulation, which assessed catalytic component (and possibly Ns) activity, was reduced in late passages. These results are consistent with acquisition by cultured UMR-106 cells of CT receptors linked to adenylate cyclase and loss of PTH and beta-adrenergic receptors. Alteration of catalytic component (and/or Ns) function may also occur after long-term culture. Since late passage cells appear dedifferentiated by chromosomal analysis and since cAMP may regulate differentiation, altered hormone-sensitive adenylate cyclase may be a marker for and a potential modulator of differentiation occurring in UMR-106 cells over long periods.