Catecholamines are major stimulants of adipose tissue metabolism. Norepinephrine and epinephrine act through three subtypes of beta-adrenoceptors (beta-AR) expressed in the adipocytes. The aim of this work was to study the mechanisms of lipid mobilization in beta1/beta2/beta3-AR triple-knockout (beta-less) mice. Glycerol and nonesterified fatty acids released from isolated adipocytes were measured as an index of lipolytic activity. There was no difference between the two genotypes for basal lipolysis and lipolytic response to corticotropin or to agents acting at the adenylyl cyclase and protein kinase A levels. The lipolytic response to norepinephrine and beta-AR agonists was blunted in beta-less mice. However, a residual low-affinity lipolytic effect was observed in the presence of catecholamines and beta3-AR agonists but not of beta1- or beta2-AR agonists. cAMP levels were increased by a beta-AR agonist in white and brown adipocytes of beta-less mice. The residual lipolytic effect was blocked by beta-AR antagonists. It was mediated neither by alpha1- or alpha2-AR nor dopaminergic, serotonergic, and histaminergic by receptors. Bioinformatic analyses do not provide evidence for a fourth beta-AR. We conclude that the residual lipolytic effect observed in beta-less mice can be attributed to an unknown Gs-protein-coupled receptor with low affinity for catecholamines.