Tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. [3H]bestim was found to bind with high affinity to mouse peritoneal macrophages (Kd 2.1 ± 0.1 nM) and thymocytes (Kd 3.1 ± 0.2 nM) and also plasma membranes isolated from these cells (Kd 18.6 ± 0.2 and 16.7 ± 0.3 nM respectively). The specific bonding of [3H]bestim with macrophages and thymocytes was inhibited by unlabeled dipeptide thymogen (L-Glu-L-Trp) (Ki 0.9 ± 0.1 and 1.1 ± 0.1 nM respectively). Treatment of the macrophages and thymocytes with trypsin led to their loss of capacity to bind [3H]bestim. Bestim at concentrations range of 0.1–1000 nM reduced the adenylate cyclase activity in macrophage and thymocyte membranes.