(-)125I-Iodocyanopindolol (125ICYP) binding to intact and ultrasonically treated human mononuclear cells (MNC) was studied. Specific binding of 125ICYP defined as the difference in binding in the presence and absence of 2 microM (+/-)-propranolol displayed in intact cells a dissociation constant (Kd) of 11.8 +/- 2.7 pM and a beta-adrenoceptor number (Rt) of 2371 +/- 154 sites/cell. This specific binding, however, still had a complex character. In broken cells a homogeneous class of binding sites with a Kd-value of 6.7 +/- 1.0 pM and a Rt-value of 883 +/- 89 sites/cell was found. When in intact cells nonspecific binding was determined with CGP-12177, a hydrophilic beta-adrenoceptor antagonist, a homogeneous class of binding sites was found with a Kd of 7.0 +/- 0.3 pM and a Rt of 1645 +/- 95 sites/cell. Inhibition curves with (+)-, (-)-, (+/-)-propranolol, timolol and CGP-12177, obtained with ultrasonically treated cells were monophasic. In intact cells propranolol and timolol not only displaced 125ICYP from its specific sites, but in an almost monophasic way also from nonspecific binding sites. CGP-12177, however, showed a clear plateau. It is concluded that in broken MNC a loss of binding sites may occur, whereas in intact cells additional binding interferes with the correct determination of specific 125ICYP binding. The latter can be reduced to a minimum by using hydrophylic ligands such as CGP-12177 to measure nonspecific binding.