Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [ 3H]diazepam being strongly inhibited by Ro5-4864 (K i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K i = 35.1 ± 18.2 μM). Binding of [ 3H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K D = 14.7 ± 1.0 nM and B max = 564 ± 75 fmoles/10 8 platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k +1 = 2.9 × 10 7 M −1 min −1, and is rapidly reversible ( t 1 2 = 2.2 min with K −1 = 0.315 min −1. K D derived from the rate constants agrees with that estimated by Scatchard analysis. K D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [ 3H]diazepam is linear with platelet number (between 0.25–2 × 10 8 platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.