A common Gbeta(3) gene polymorphism (C825T) influences G protein receptor-mediated signal transduction. We investigated whether this polymorphism influences lipolysis in isolated subcutaneous fat cells from 114 healthy obese subjects. The Gbeta(3) protein content was markedly decreased in adipocytes of TT carriers, but the alternatively spliced short form of Gbeta(3) previously shown in platelets of 825T carriers was not detected. Fat cells of TT carriers showed a significant 10-fold decrease in the half-maximum effective concentration of agonists selective for lipolytic beta(1)- and beta(2)-adrenoceptors as well as for the antilipolytic alpha(2)A-adrenoceptor. In TT carriers, maximum beta-adrenoceptor agonist-stimulated lipolysis was decreased, but the maximum antilipolytic effect of alpha(2)-adrenoceptors was less marked. Norepinephrine induced adipocyte lipolysis and circulating fasting levels of free fatty acids and glycerol were reduced by half in TT carriers. The polymorphism did not influence the adipocyte content of alpha(2)A-adrenoceptors, beta(2)-adrenoceptors, Galpha(i), or Galpha(s). In conclusion, the C825T variant of Gbeta(3) influences lipolysis. Adipocytes of TT carriers have a lower Gbeta(3) protein content and a decreased function of native G(s)- as well as G(i)-coupled adrenoceptors, which reduces the lipolytic effect of catecholamines. These data differ from those obtained in other cell systems that have shown increased expression of an alternative spliced Gbeta(3) variant and enhanced G protein signaling in 825T carriers, indicating that the polymorphism has cell type-specific effects that may be of importance for type 2 diabetes and other insulin-resistant conditions.