Previous studies have shown that beta 3-adrenergic receptors, in contrast to the beta 1 and beta 2 subtypes, do not undergo desensitization following short term activation (minutes) with agonists. Longer activation (hours) has been shown to induce desensitization of the beta 3-adrenergic receptor in some, but not all, cases, suggesting that cell- or species-specific mechanisms may be involved. We investigated the contribution of the cell type to the pattern of beta 3-adrenergic receptor long term desensitization by studying, in parallel, two cell lines (Chinese hamster fibroblasts and murine Ltk- cells) expressing the human beta 3-adrenergic receptor. Sustained agonist-promoted down-regulation of the beta 3-adrenergic receptor could be induced in Ltk- cells, whereas only a transient and weak reduction of receptor number was observed in Chinese hamster fibroblasts. The half-life of the beta 3-adrenergic receptor was not affected by the agonist activation in either cell line, indicating that in contrast to the beta 2-adrenergic receptor, degradation of preexisting receptor protein does not contribute to down-regulation. Sustained reduction of receptor RNA levels, monitored by reverse transcriptase polymerase chain reaction, was exclusively shown in Ltk- cells and probably accounted for most of the observed down-regulation. Differences in the ability of the receptor to stimulate adenylyl cyclase activity in the two cell lines may be responsible for the distinct patterns of beta 3-adrenergic receptor down-regulation.