In vitro function of fresh platelets and platelets stored for 24 hours at 4 C and 22 C was studied using the release of14C‐5‐hydroxytryptamine (5‐HT), adenine nucleotides and calcium following stimulation with thrombin. Storage of platelets did not affect14C‐5‐HT uptake capability, adenine nucleotide content or adenine nucleotide release. Platelets stored in 4 C varied from fresh platelets and 22 C‐stored platelets with respect to 5‐HT and calcium release. For measurement of 5‐HT release, different thrombin‐to‐platelet concentrations were used. At thrombin‐to‐platelet concentrations of 0.013 to 0.02 U/108platelets, 4 C‐stored platelets released more 5‐HT than did fresh platelets. Differences between 4 C‐stored platelets and 22 C stored platelets were observed over a wider range of thrombin‐to‐plate‐lets concentrations. The 4 C‐stored platelets released significantly greater amounts of 5‐HT at thrombin‐to‐platelet concentrations of 0.008‐0.02 L/ml. Calcium release, measured with the calcium electrode, was studied at high (1.0 L/ml) and low (0.06 U/ml) thrombin concentrations and at a platelet concentration of 8 × 108platelets/ml. At the high thrombin concentration, the amount of calcium released was the same for all platelets studied. At the low thrombin concentration, platelets stored at 4 C released a greater amount of calcium than did either fresh platelets or platelets stored at 22 C. The results obtained with different thrombin‐to‐platelet concentrations suggest that there is only a narrow range of thrombin concentrations per platelet at which the subtle differences between platelet populations may be detected. The range may reflect the threshold concentration of thrombin that is required to initiate a release reaction. The apparently lower thrombin threshold required by 4 C platelets to undergo a release reaction may be a factor contributing to their reduced