PKC is a critical effector of plasma membrane dynamics, yet the mechanism and isoform-specific role of PKC are poorly understood. We recently showed that the phorbol ester PMA (100 nM) induces prompt activation of the novel isoform PKCε followed by late activation of the conventional isoform PKCα in T84 intestinal epithelia. PMA also elicited biphasic effects on endocytosis, characterized by an initial stimulatory phase followed by an inhibitory phase. Activation of PKCε was shown to be responsible for stimulation of basolateral endocytosis, but the role of PKCα was not defined. Here, we used detailed time-course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKCα on basolateral endocytosis. Inhibition of PKCα by the selective conventional PKC inhibitor Gö-6976 (5 μM) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis measured by FITC-dextran uptake. The PKCε-selective agonist carbachol (100 μM) induced prolonged stimulation of endocytosis devoid of an inhibitory phase. Actin disassembly caused by PMA was completely blocked by Gö-6850 but not by Gö-6976, implicating PKCε as the key isoform responsible for actin disruption. The Ca2+agonist thapsigargin (5 μM) induced early activation of PKCα when added simultaneously with PMA. This early activation of PKCα blocked the ability of PMA to remodel basolateral F-actin and abolished the stimulatory phase of basolateral endocytosis. Activation of PKCα stabilizes F-actin and thereby opposes the effect of PKCε on membrane remodeling in T84 cells.