Cardiac β-adrenoceptors are assumed to play a key role in chronic heart failure. Although several radioligands labeled with 1 1 C or 1 8 F have been synthesized for imaging purposes with positron emission tomography (PET), so far no optimal ligands are available to image cardiac β-adrenoceptors using single photon emission tomography (SPECT). In the present study, we characterized four new synthesized analogues of the nonselective β-adrenoceptor antagonist 4-(3-t-butylamino-2-hydroxypropoxy)-benzimidazol-2-one (CGP12177) and one analogue of the nonselective β-adrenoceptor antagonist penbutolol. Using classical in vitro displacement studies with left ventricular tissue of New Zealand White rabbits and [ 1 2 5 I]iodocyanopindolol as a radioligand, binding affinity to the receptor was determined. From the four analogues, only (2 S,2 E)- [4-(3 -(1 ,1 -dimethyl-3 -Iodo-2 propenylamino)-2 -hydroxypropoxy)]-be nzimidazol-2-one proved to have a high affinity, with K i = 1.25 ± 0.09 nM, n = 3. The other analogues showed relatively low affinity, with K i -values > 1 nM. The analogue of penbutolol ((S)-(-)-[1-(2-Iodophenoxy)]-3 -(tert-butylamino)-2 -propanol) also showed a K i value of 0.64 ± 0.26 nM, n = 3. Subsequently, (2 S,2 E)-[4-(3 -(1 ,1 -dimethyl-3 -Iodo-2 propenylamino)-2 -hydroxy propoxy)]-benzimidazol-2-one and (S)-(-)-[1-(2-Iodophenoxy)]-3 -(tert-butylamino)-2 -propanol were radioactively labeled with 1 2 3 I to study their biodistribution in New Zealand White rabbits and to determine specific binding. Significant uptake was observed in both lungs and left ventricles. However, both compounds showed high nonspecific binding in vivo because uptake of the radioligand could not be inhibited by preinjection of different (selective-and nonselective-adrenoceptor antagonists and hydrophilic and lipophilic antagonists) antagonists. In conclusion, although two analogues showed reasonable affinity in vitro for the receptor, their binding in vivo proved to be largely nonspecific, suggesting that these two compounds are unsuitable for imaging purposes. However, because marked differences in affinity for the receptor were observed with only little structural changes between compounds, the present results offer future perspectives for the synthesis of a more specific radioligand.