White fat adipocytes have a Vm, membrane potential, of -30 mV under physiological ionic conditions which is shown to be mainly due to Cl- permeability (1). Flux and imaging studies demonstrate that their intracellular Ca2+ concentration, [Ca2+]i is influenced by a Ca2+ window current flowing through spontaneously active L-type voltage-gated Ca2+ channels (2,3); an observation consistent with their depolarised resting membrane potential. Given these facts, we have investigated if changes in extracellular Cl- concentration, [Cl-]o, can affect [Ca2+]i via Vm mediated changes in voltage-gated Ca2+ influx. White fat adipocytes were isolated from subcutaneous fat of male CD-1 mice. After plating onto coverslips, [Ca2+]i was imaged using convention epifluorescent microscopy as described (1). Cells were continuously perifused at 28°C in a Hanks salt solution, control, with a [Cl-]o of 152 mM. The effect of serial equimolar substitution of bath NaCl with either Na gluconate or Na glutamate to decrease [Cl-]o on [Ca2+]i was explored. Data are given as mean±S.E.M. Statistical significance, p<0.05, was determined by ANOVA with Holm-Sidak’s multiple comparison test. Substitution of NaCl with Na gluconate (n = 39) to give 113 mM [Cl-]o did not affect [Ca2+]i. However, further reduction of [Cl-]o to 53 mM, and then to 18 mM significantly decreased [Ca2+]i from 127±9 nM to 117±8 nM and then to 113±7 nM respectively. [Ca2+]i only partly recovered to 115±7 nM on return to normal control Hanks. Repetition of these experiments but with Na glutamate (n = 32) instead also decreased [Ca2+]i: substitution of [Cl-]o to 113 significantly decreased [Ca2+]i from 165±9 nM to 142±9 nM after which it increased back to 150±9 nM at 53 mM. By 18 mM [Cl-]o [Ca2+]i had returned to control levels. To explore the mechanistic differences in the [Ca2+]i response to the different anions we looked at their physical chemistry. Since gluconate is reported to chelate Ca2+ (4) we analysed the free Ca2+ of our solutions. Gluconate, but not glutamate, was found to chelate free Ca2+, the former with a Kd of 33.1±0.7 mM (n=3). So the effect of gluconate substitution was repeated but with [Ca2+]o titrated back to that measured in control Hanks solution: 2.7±0.3 mM (n=3). Under these conditions, substitution of bath Cl- with gluconate had no effect on [Ca2+]i. Possible mechanisms for the effects with glutamate were not forthcoming and have yet to be investigated. In conclusion, the physiochemical properties of the anion used for chloride substitution confounded measurement of [Ca2+]i and attempts to gate voltage-dependent Ca2+ influx in white fat adipocytes. Consquently, the free [Ca2+] of gluconate containing salt solutions should always be checked. Overall, changes in [Cl-]o ,and so Vm, do not appear to affect voltage-gated Ca2+ entry and [Ca2+]i of white fat adipocytes.