When C6-rat glioma cells were incubated for 20 min with beta-adrenoceptor agonists, a part of the beta-adrenoceptors was localized in light density vesicles. These receptors have the same affinity for dihydroalprenolol as plasma membrane receptors but have a lowered affinity for agonists as well as for the hydrophilic beta-adrenoceptor antagonist CGP-12177. The affinity of these vesicular receptors for a variety of hydrophobic beta-blockers as well as for the hydrophilic beta-blocker timolol is compared with that of plasma membrane receptors. None of the ligands investigated showed any difference in affinity but CGP-12177. Both, introducing a lipophilic side chain into CGP-12177 or altering the benzimidazol-2-one ring system of CGP-12177 led to an increase in the affinity of the vesicular receptors. Since in the presence of the pore-forming agent alamethicin the same affinity was determined for vesicular receptors as for those located on the plasma membrane, it is concluded that the apparent low affinity of the vesicular receptors in the absence of alamethicin is caused by a membrane barrier. The lower affinity of the vesicular receptors for agonists was only slightly increased by alamethicin.