The plasma membrane potential (Vm) drives many physiological processes; its dysregulation often underlies functional pathologies. K+ is widely believed to be the predominant ion that controls Vm for many cell types. Although, the plasma membrane of primary white fat adipocytes contains K+ channel conductances (Lee and Pappone 1997), whether they control its Vm is unknown. We have investigated the ionic species involved in the control of Vm of primary and differentiated 3T3-L1 adipocytes. Since insulin and β-adrenoceptors are key regulators of adipocyte function we also explored their effect on adipocyte Vm. Primary adipocytes were isolated via enzymatic dispersion of rat (250-340g) epididymal fat pads as described by (Rodbell 1964). 3T3-L1 adipocytes were prepared as described by (Mehra, Macdonald et al. 2007). We measured Vm with the perforated patch clamp technique at 32oC. Values are given as mean± S.E.M. and are compared with Friedman’s test or Wilcoxon sign test as appropriate. The Vm of primary and 3T3-L1 adipocytes were -34.4±1.5mV (n=68) and -28.5±1.2mV (n=88) respectively. Elevation of extracellular K+ from 5.6 to 50mM, by equimolar substitution of bath Na+, had no significant effect on the Vm of either type of adipocyte (n=8-11). As a positive methodological control, similar experiments performed on primary mouse beta cells demonstrated that the same change in extracellular K+ reversibly depolarised their Vm from -71.7±1.8 to -20.8±2.4mV (n=8; P<0.001); a result similar to that reported by others (Atwater, Ribalet et al. 1978). We next investigated the effect of Cl- on adipocyte Vm. Reduction of extracellular Cl- from 138 to 5mM, by equimolar substitution with gluconate, significantly depolarised the Vm of both primary and 3T3-L1 adipocytes to -9±4mV (n=9; P<0.005) and -9.6±3.7mV (n=12; P<0.005) respectively. Equimolar substitution of extracellular Na+. from 148 to 10mM with N-methl-D-glucamine, significantly hyperpolarised the Vm of both primary and 3T3-L1 adipocytes to -39±5mV (n=7; P<0.05) and -49±4.7mV (n=7; P<0.05) respectively. Neither insulin (100nM) or the β-adrenoceptor agonist isoprenaline (10µM) significantly changed adipocyte Vm (n=3-12). Our data indicates that the Vm of primary and differentiated 3T3-L1 adipocytes is predominantly controlled by Cl-, and not K+, with a minor role for Na+. Furthermore, we show that the functional effects of insulin and β-adrenoceptor stimulation on adipocytes are unlikely to be modulated by alterations in membrane potential.