Adenosine acting at adenosine A1 receptors is considered to be one major regulator of adipose tissue physiology. We have examined the role of adenosine and its interactions with insulin in adipose tissue by using A1R knock out (-/-) mice. Removal of endogenous adenosine with adenosine deaminase caused lipolysis in A1R (+/+), but not A1R (-/-) adipocytes. The adenosine analogue, 2-chloroadenosine, inhibited noradrenaline-stimulated lipolysis and cAMP accumulation in A1R (+/+), but not in A1R (-/-) adipocytes. Insulin reduces lipolysis and cAMP via another mechanism than adenosine and acted additively, but not synergistically, with adenosine. Plasma levels of free fatty acids, glycerol and triglycerides were significantly lower in A1R (+/+) than in A1R (-/-) mice after administration of an adenosine analogue. 2-chloroadenosine induced lipogenesis in presence of insulin in A1R (+/+), but not in A1R (-/-) adipocytes. There were no changes in mRNA levels for several genes involved in fat synthesis in adipose tissue between genotypes. Body weight was similar in young A1R (+/+) and A1R (-/-) mice, but old male A1R (-/-) mice were heavier than wild type controls. In conclusion, adenosine inhibits lipolysis via the adenosine A1 receptor and other adenosine receptors play no significant role. Adenosine and insulin mediate additive but not synergistic antilipolytic effects and 2-chloroadenosine stimulates lipogenesis via adenosine A1 receptors. Thus deletion of adenosine A1 receptors should increase lipolysis and decrease lipogenesis, but in fact an increased fat mass was observed, indicating that other actions of adenosine A1 receptors, possibly outside adipose tissue, are also important.