Protein kinase C (PKC), a multi-gene family of enzymes, plays key roles in the regulation of signal transduction and cellular function in various cell types. Intracellular localization and trafficking of these individual PKC isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated at downstream of Gq-protein coupled receptor (GqPCR) signaling and recent reports suggested that different type of GqPCRs would activate different isoforms of PKC (classic, novel and atypical PKCs). However, the knowledge of isoform specific activation of PKC by GqPCRs is limited. αλπηα1-adrenergic receptor (αλπηα1-AR) is the one of the GqPCR strongly expressed in the heart, vascular system and urinary bladder. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by αλπηα1-adrenergic stimulation. Rat PKCαλπηα, βeταI, βeταII, δeλτα, eπσιλoν and ζeτα were fused with GFP at C-term and were co-transfected with human αλπηα1-AR to HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by αλπηα1-AR stimulation using phenylephrine were observed by confocal microscope. Before stimulation, GFP-PKCs were localized at cytosolic region. αλπηα1-AR stimulation strongly and rapidly translocated classical PKCαλπηα (< 30s) to the plasma membrane (PM), with PKCαλπηα diffusing back into the cytosol within 5 min. αλπηα1-AR stimulation rapidly translocated classical PKC βeταI and II to the PM (<30s), with sustained membrane localization. Novel PKCeπσιλoν, but not δeλτα were translocated by αλπηα1-AR stimulation to the PM (<30s) and its membrane localization was also sustained. αλπηα1-AR stimulation did not caused DAG-insensitive atypical PKCζeτα translocation. Our data suggests that PKCαλπηα, PKCβeτα and PKCeπσιλoν activation may underlie αλπηα1-AR function. Our data suggest that PKCβeτα and PKCeπσιλoν may be particular important during chronic αλπηα1-AR and cardiovascular disease.