This study characterizes the mouse β3a‐adrenoceptor (AR) and the splice variant of the β3‐AR (β3b‐AR) expressed in Chinese hamster ovary cells (CHO‐K1).Stable clones with high (∼1200), medium (∼500) or low receptor expression (∼100 fmol mg protein−1) were determined by saturation binding with [125I]‐(−)‐cyanopindolol. Competition binding studies showed no significant differences in affinity of β‐AR ligands for either receptor.Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The β3‐AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non‐conventional partial β‐AR agonist CGP12177 and the β‐AR agonist (−)‐isoprenaline caused concentration‐dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration‐dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor.PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the β3b‐AR but not in cells expressing the β3a‐AR at all levels of receptor expression.CL316243 increased Erk1/2 phosphorylation with pEC50 values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3Kγ inhibitor), PP2 (c‐Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor).The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both β3a‐ or β3b‐AR transfected cells.These results suggest that in CHO‐K1 cells, the β3b‐AR, can couple to both Gs and Gi to stimulate and inhibit cyclic AMP production respectively, while the β3a‐AR, couples solely to Gs to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to Gi or the generation of cyclic AMP.British Journal of Pharmacology (2002) 135, 1903–1914; doi:10.1038/sj.bjp.0704654