The β3-adrenergic receptor (β₃-AR) is one of three G-protein coupled receptors which mediate the physiological effects of the catecholamines adrenaline and noradrenaline. In common with the β₁– and β₂-adrenoceptors, the β₃-AR mediates its effects through activation of stimulatory (G_s) G-proteins to increase cellular levels of cAMP (Arch, 2001). However, unlike these closely related receptors, the β₃-AR is resistant to short term desensitisation and internalisation following agonist exposure (Strosberg, 1997). Here, we have investigated the effect of agonist exposure on the diffusion of the β₃-AR in the membrane microdomains of individual cells using fluorescence correlation spectroscopy (FCS). The β₃-AR was fused via its C-terminus to eGFP in the vector pcDNA3.1. This vector was transfected transiently into Chinese hamster ovary cells (CHO-β3GFP cells) using Lipofectamine, according to manufacturer’s instructions. Cells were exposed to the β₃-AR agonist isoprenaline (10^-5M) or the antagonist ICI118551 (10^-5M) at 37°C as indicated, prior to FCS measurements being taken on the upper cell membrane, as described previously (Briddon et al., 2004). Measurements were taken for 2x15s following a 15s pre-bleach, and autocorrelation data anlysed in Zeiss AIM software. Autocorrelation curves from FCS measurements on the upper cell membrane of CHO-β3GFP cells were best fit assuming two diffusion components (τ_D1 = 396±17μs and τ_D2 = 82.6±6.4ms, n=52). Of the total diffusing receptor population, approximately 60% was present as the faster-diffusing component (Particl numbers for τ_D1 = 37.1±4.6 and for τ_D2 = 53.0 ± 6.4 μm^-2). As shown in Table 1, exposure to isoprenaline for 60 min caused an increase in τ_D1, indicating a slowing of receptor diffusion. This was preceeded by an increase in the number of receptors showing that slower diffusion time. In contrast, antagonist exposure had no significant effect on receptor diffusion. These results indicate that, whilst the β₃-AR may not internalise readily, agonist stimulation causes a decrease in the diffusivity of the receptor within the cell membrane. This slowing could be a result of receptor aggregation, or possibly agonist-induced movement of receptor between differing membrane compartments.