Ubiquitination, proteasome, caveolae and endosomes have been implicated in controlling protein kinase C alpha (PKC alpha) down-regulation. However, the molecular mechanism remained obscure. Here we show that endosomes and proteasome cooperate in phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA)-induced down-regulation of PKC alpha. We show that following TPA treatment and translocation to the plasma membrane, PKC alpha undergoes multimonoubiquitination prior to its degradation by the proteasome. However, to reach the proteasome, PKC alpha must travel through the endocytic system from early to late endosomes. This route requires functional endosomes, whereby endosomal alkalinization, or ablation, abrogates completely PKC alpha degradation maintaining the enzyme at the plasma membrane. This route also depends on synaptotagmin (Syt) II and the Rab7 GTPase, whereby Syt II knock-down or expression of the GDP-locked Rab7 inactive mutant prevents PKC alpha degradation. We further show that proteasome plays a dual role, where an active proteasome is required for deubiquitination of PKC alpha, a step crucial to prevent PKC alpha targeting to the endocytic recycling compartment. Finally, we show that the association with rafts-localized cell surface proteins that internalize in a clathrin-independent fashion is necessary to allow the trafficking of PKC alpha from the plasma membrane to the proteasome, its ultimate degradation station.