Chlorethylclonidine (CEC) inactivation has been used as one criterion to subclassify the alpha1-adrenoceptors (AR); however, the extent of CEC inactivation can vary depending on the CEC treatment. By constructing the FLAG-tagged (N-terminus) and green fluorescent protein (GFP)-fused (C-terminus) alpha1-ARs, we have determined the relationship between CEC sensitivity and the cellular localization of alpha1-AR subtypes using COS-7 cells. In GFP-expressing cells, flow cytometry analysis with anti-FLAG N-terminus antibody detected strong fluorescent signals in most of alpha1B-AR-expressing cells, but low signals in alpha1A-AR-expressing cells. Further examination with confocal microscopy showed that fluorescent signals densely localized intra-cellularly in alpha1A-AR-expressing cells, while most of alpha1B-AR localized on the cell surface. Furthermore, radioligand binding studies with [125I]HEAT showed that CEC (10 microM) treatment of intact cells inactivated approximately 30-40% of alpha1A-AR and >90% of alpha1B-AR, while the CEC treatment of membrane preparations resulted in >80% decrease in the alpha1A-AR density and >90% of alpha1B-AR density, respectively. The results showed that the hydrophilic alkylating agent CEC inactivated only alpha1-AR on the cell surface irrespective of its subtype, and that the subtype-specific sorting is a major determinant for CEC inactivation of alpha1-AR. Subtype-specific cellular localization suggests a new class of functional properties that may explain the signal and functional diversity of homologous alpha1-AR (as well as other G protein-coupled receptors) subtypes.