The membrane potential of human platelets, and the role of this potential in platelet aggregation, was assessed using the non-covalent, fluorescent probe DiS-C3-5. High K+ and Gramicidin depolarised the cells, whereas valinomycin in standard (4 mMK+) solution produced a hyperpolarisation. Very small changes in potential were observed when choline Cl replaced NaCl. These findings indicate that platelets possess a relatively K+-permselective membrane. The resting potential calculated from the “valinomycin null point” (the K+ concentration gradient at which valinomycin did not change the potential) was approximately – 60 mV. Other factors that contribute to the platelet membrane potential include a significant Cl− permeability, demonstrated by replacing Cl− with methylsulphate, and an electrogenic Na+ pump, demonstrated using strophanthidin. Little or no change in potential was observed upon addition of ADP, collagen, U44069 or thrombin. Neither strong depolarisation with high K+ or gramicidin nor hyperpolarisation with valinomycin induced platelet aggregation or altered platelet responses to agonists. It is concluded that the information transduction mechanisms involved in platelet activation do not include changes in platelet membrane potential.