1. [3H]-bradykinin was used to characterize the bradykinin receptors associated with canine cultured tracheal smooth muscle cells (TSMCs). Receptor binding assay showed that TSMCs had specific, saturable, high-affinity binding sites for [3H]-bradykinin. 2. The specific [3H]-bradykinin binding increased linearly with increasing cell concentrations. The equilibrium for association of [3H]-bradykinin with the bradykinin receptors was attained within 2 h at 4 degrees C and 1 h at room temperature, respectively. 3. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 2.5 +/- 0.3 nM and a maximum receptor density (Bmax) of 25.1 +/- 0.3 fmol mg-1 protein. The Hill coefficient for [3H]-bradykinin binding was 1.00 +/- 0.02. The association (K1) and dissociation (K-1) rate constants were (8.67 +/- 2.60) x 10(6) M-1 min-1 and 0.024 +/- 0.005 min-1, respectively. KD, calculated from the ratio of K-1 and K1 was 2.8 +/- 0.5 nM, a value close to that of KD calculated from Scatchard plots of binding isotherms. 4. The B1 receptor selective agonist, (des-Arg9-bradykinin, 0.1 nM-10 microM) and antagonist ([Leu8, des-Arg9]-bradykinin, 0.1 nM-10 microM) did not did not inhibit the [3H]-bradykinin binding to TSMCs, which excludes the presence of B1 receptors in canine TSMCs. 5. The specific binding of [3H]-bradykinin to canine TSMCs was inhibited by B2 receptor selective antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oicl-bradykinin, Hoe 140, 0.1 nM-10 micro M and [D-Arg0, Hyp3,Thi5,8, D-Phe7-bradykinin, 0.1 nM-10 micro M) and agonists (bradykinin and kallidin, 0.1 nM-10 micro M) with a best fit by a one-binding site model. The order of potency for the inhibition of [3H]-bradykinin binding was kallidin = bradykinin = Hoe 140> [D-Arg0, Hyp3, Thi5,8, D-Phel-bradykinin.6. Preincubation of TSMCs with forskolin for 24 h led to an up-regulation of B2 receptors, increasing in Bmax from 25.1 +/- 0.3 to 218 +/- 24 fmol mg-1 protein without changing the KD values. [3H]-bradykinin binding to TSMCs was inhibited by the B2 receptor selective antagonists and agonists, but not by the B1 receptor selective reagents. The up-regulation of the B2 receptor by forskolin was mediated through protein synthesis, since cycloheximide blocked this response.7 It is concluded that the pharmacological characteristics of the bradykinin receptors in canine cultured TSMCs are primarily of the B2 receptor subtype.