The presence of muscarinic receptors in intact and culture bovine adrenomedullary chromaffin cells was investigated by a binding assay using the antagonist-specific high affinity ligand, [(3)H] quinuclidinyl benzilate [((,3)H)-QNB]. The kinetics of [(3)H]-QNB binding to partially purified, mechanically isolated intact bovine chromaffin cells was examinde. The rate constants at 37Degree C for association and dissociation of the [(3)H]-QNB-receptor complex in intact cells were 1.2 x10(8) M(-1) min(-1) and 1.28 x 10(-2) min(-1), respectively. The dissociation equilibrium constant (Kd) can be derived from dissociation and association rate constants (k-1/k+1). The Kd value was calculated to be 0.106 nM. The present study demonstrated that collagenase at 0.1% or higher concentrations decreased the specific binding of [(3)H]-QNB to muscarinic receptors in mechanically isolated intact bovine chromaffin cells. Scatchare analysis combined with computer program "LIGAND" was used for characterization of the specific [(3)H]-QNB binding. It was found that partially and Renografin purified, mechanically isolated intact bovine chromaffin cells had a single, high affinity class of binding sites with a Kd of 0.12 (+or-)0.02 nM and 0.09 (+or-) 0.03 nM and a Bmax of 3.1 (+or-) 0.6 fmol/10(3) cells and 0.45 (+or-) 0.16 fmol/10(3) cells, respectively. In addition, partially and Renografin purified, enzymatically (0.15% collagenase) isolated intact bobine chromaffin cells had a single, high affinity class of binding sites with a Kd of 0.14 (+or-) 0.06 nM and 0.02 (+or-) 0.004 nM and a Bmax of 0.021 (+or-) 0.0007 fmol/10(3) cells and 0.01 (+or-) 0.003 fmol/10(3) cells, respectively. Interestingly the 7-day culture of bovine chromaffin cells exhibited a two class of binding sites with a Kd of 0.01 (+or-) 0.003 nM and a Bmax of 0.006 (+or-) 0.001 fmol/10(3) cells for the high affinity sits and a Kd of 17.0 (+or-) 6.0 nM and a Bmax of 0.13 (+or-) 0.06 fmol/10(3) cells for the low affinilty site, respectively. These data demonstrate that 0.15% collagenase alters the receptor density (Bmax) fo mAChR in intact and culture bovine adrenomedullary chromaffin cells but the Kd remains unchanged.