This study examines signaling pathways activated by the mouse beta3-adrenoceptor (AR) expressed in Chinese hamster ovary cells at high (CHObeta3H) or low (CHObeta3L) levels. Functional responses included extracellular acidification rate (ECAR), cyclic AMP accumulation, and p38 MAPK or Erk1/2 phosphorylation. (-)-Isoproterenol and the beta3-AR agonist CL316243 caused concentration-dependent increases in cAMP accumulation and ECAR in CHObeta3H and CHObeta3L cells. For cAMP accumulation, the beta3-AR ligand SR59230A was a partial agonist in CHObeta3H and an antagonist in CHObeta3L cells but for ECAR was an agonist at both expression levels. This suggested that SR59230A which is normally regarded as an antagonist can selectively activate pathways leading to ECAR. Examination of the pathways stimulated by (-)-isoproterenol, CL316243 and SR59230A for both ECAR and cAMP accumulation suggested that the cAMP pathway predominates in CHObeta3H cells while p38 MAPK is a major contributor to ECAR in CHObeta3L cells and was the sole contributor to responses to SR59230A. Western blots of p38 MAPK and Erk1/2 phosphorylation confirmed that MAPKs are activated in CHObeta3H and CHObeta3L cells by CL316243 and SR59230A but that SR59230A has much higher efficacy. In addition, p38 MAPK phosphorylation displayed differences in drug potency and efficacy between CHObeta3H and CHObeta3L cells related to inhibition of the response by cAMP. Thus CL316243 and SR59230A display reversed orders of efficacy for cAMP accumulation compared to Erk1/2 and p38 MAPK phosphorylation providing a strong indication of ligand-directed signaling.