Brown adipose tissue (BAT) is the main site of adaptive thermogenesis, and its function is mostly regulated by the sympathetic nervous system and the β‐adrenergic receptor signaling pathway. To evaluate the independent roles played by β1 and β2 isoforms in energy homeostasis, mice with targeted disruption of either one of these genes were studied. While the KOβ1 mice failed to increase resting energy expenditure (REE) and developed hypothermia during cold exposure, they also exhibited decreased interscapular BAT thermal response during norepinephrine or dobutaine infusion. In contrast, KOβ2 mice behaved normally under these conditions. When placed on a high fat diet (24% fat) for 21 weeks, both KOβ1 and KOβ2 mice doubled their caloric intake, body weight gain and body fat content, not different from wild type animals, but failed to increase REE. In conclusion, our data indicate that the β1 signaling pathway is critical in cold‐induced thermogenesis whereas both β1 and β2 pathways play a role in the thermogenesis induced by high‐fat feeding. Supported by FAPESP.