Selective inactivation of alpha1B-adrenoceptor (AR) by the site-directed alkylating agent chlorethylclonidine (CEC) has been used as one of major pharmacological criteria to subclassify alpha1-AR; however, the mechanism for the differential CEC sensitivity of the two subtypes is uncertain, and the extent of CEC inactivation varies depending on the treatment employed. In this study, we examined the correlation between the subcellular localization of alpha1-AR subtypes (alpha1A and alpha1B) and CEC sensitivity. Constructing alpha1-AR tagged with the FLAG epitope at the amino terminus and/or green fluorescent protein (GFP) at the carboxyl terminus, we examined the subcellular distribution of alpha1-ARs expressed in COS-7 cells. Flow cytometry analysis showed that most populations of GFP-expressing alpha1B-AR cells, but very few GFP-expressing alpha1A-AR cells, were detected by the anti-amino terminus antibodies. The immunocytochemical and GFP-fluorescence confocal micrographs showed that alpha1A-ARs predominantly localize intracellularly, whereas alpha1B-ARs localize on the cell surface. Furthermore, CEC (10 microM) treatment of intact cells resulted in an inactivation of approximately 42% of alpha1A-ARs and 93% of alpha1B-ARs, whereas treatment of the membrane preparations resulted in an inactivation of approximately 83% of alpha1A-ARs and 88% of alpha1B-ARs, respectively. Together, the results showed that a hydrophilic alkylating agent CEC preferentially inactivates alpha1-AR on the cell surface irrespective of its subtype, and that the subtype-specific subcellular localization rather than the receptor structure is a major determinant for CEC inactivation of alpha1-AR. Subtype-specific subcellular localization suggests an additional class of functional properties that provide new insight into drug action.