beta3-Adrenergic receptors (beta3-ARs) are expressed predominantly on white and brown adipocytes, and acute treatment of mice with CL 316,243, a potent and highly selective beta3-AR agonist, produces a 2-fold increase in energy expenditure, a 50-100-fold increase in insulin levels, and a 40-50% reduction in food intake. Recently, we generated gene knockout mice lacking functional beta3-ARs and demonstrated that each of these responses were mediated exclusively by beta3-ARs. However, the tissue site responsible for producing these actions is unknown. In the present study, genetically engineered mice were created in which beta3-ARs are expressed exclusively in white and brown adipocytes (WAT+BAT-mice), or in brown adipocytes only (BAT-mice). This was accomplished by injecting tissue-specific beta3-AR transgenic constructs into mouse zygotes homozygous for the beta3-AR knockout allele. Control, knockout, WAT+BAT, and BAT-mice were then treated acutely with CL, and the effects on various parameters were assessed. As previously observed, all effects of CL were completely absent in gene knockout mice lacking beta3-ARs. The effects on O2 consumption, insulin secretion, and food intake were completely rescued with transgenic re-expression of beta3-ARs in white and brown adipocytes (WAT+BAT-mice), demonstrating that each of these responses is mediated exclusively by beta3-ARs in white and/or brown adipocytes, and that beta3-ARs in other tissue sites were not required. Importantly, transgenic re-expression of beta3-ARs in brown adipocytes only (BAT-mice) failed to rescue, in any way, CL-mediated effects on insulin levels and food intake and only minimally restored effects on oxygen consumption, indicating that any effect on insulin secretion and food intake, and a full stimulation of oxygen consumption required the presence of beta3-ARs in white adipocytes. The mechanisms by which beta3-AR agonist stimulation of white adipocytes produces these responses are unknown but may involve novel mediators not previously known to effect these processes.