Brown adipose tissue (BAT) is currently considered as a target to combat obesity and diabetes in humans. BAT is densely innervated by the sympathetic nervous system (SNS) and can be stimulated by β-adrenergic agonists, at least in animals. However, the exact role of the β-adrenergic part of the SNS in BAT activation in humans is not known yet. In this study, we measured BAT activity by 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) positron emission tomography/computed tomography imaging in 10 lean men during systemic infusion of the nonselective β-agonist isoprenaline (ISO) and compared this with cold-activated BAT activity. ISO successfully mimicked sympathetic stimulation as shown by increased cardiovascular and metabolic activity. Energy expenditure increased to similar levels as during cold exposure. Surprisingly, BAT was not activated during β-adrenergic stimulation. We next examined whether the high plasma free fatty acid (FFA) levels induced by ISO competed with glucose ([¹⁸F]FDG) uptake in BAT locations by blocking lipolysis with acipimox (ACI). ACI successfully lowered plasma FFA, but did not increase [¹⁸F]FDG-uptake in BAT. We therefore conclude that systemic nonselective β-adrenergic stimulation by ISO at concentrations that increase energy expenditure to the same extent as cold exposure does not activate BAT in humans, indicating that other tissues are responsible for the increased β-adrenergic thermogenesis.