Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated downstream of G(q)-protein coupled receptor (G(q)PCR) signaling and translocate to various cellular compartments including plasma membrane (PM). Recent reports suggested that different types of G(q)PCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each G(q)PCR is limited. alpha(1)-Adrenoceptor (alpha(1)-AR) is one of the G(q)PCRs highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by alpha(1)-AR stimulation (alpha(1)-ARS). Rat PKC alpha, beta I, beta II, delta, epsilon and zeta fused with GFP at C-term were co-transfected with human alpha(1A)-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by alpha(1)-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. alpha(1)-ARS strongly and rapidly translocated a classical PKC (cPKC), PKC alpha, (<30 s) to PM, with PKC alpha returning diffusively into the cytosol within 5 min. alpha(1)-ARS rapidly translocated other cPKCs, PKC beta I and PKC beta II, to the PM (<30 s), with sustained membrane localization. One novel PKC (nPKC), PKC epsilon, but not another nPKC, PKCS, was translocated by alpha(1)-AR stimulation to the PM (<30 s) and its membrane localization was also sustained. Finally, alpha(1)-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKC zeta translocation. Our data suggest that PKC alpha, beta and epsilon activation may underlie physiological and pathophysiological responses of alpha(1)-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system. (C) 2015 Elsevier Inc. All rights reserved.