The involvement of PKC, the isoforms of which are categorized into three subtypes: conventional (α, βI, βII, and γ), novel [δ, ϵ, η, and μ (also known as PKD),θ], and atypical (ζ and ι/λ), in the regulation of endothelial monolayer integrity is well documented. However, isoform activity varies among different cell types. Our goal was to reveal isoform-specific PKC activity in the microvascular endothelium in response to phorbol 12-myristate 13-acetate (PMA) and diacylglycerol (DAG). Isoform activity was demonstrated by cytosol-to-membrane translocation after PMA treatment and phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein after PMA and DAG treatment. Specific isoforms were inhibited by using both antisense oligonucleotides and pharmacological agents. The data showed partial cytosol-to-membrane translocation of isoforms α, βI, and ϵ and complete translocation of PKCδ and PKD in response to PMA. Furthermore, antisense treatment and pharmacological studies indicated that the novel isoform PKCδ and PKD are both required for PMA- and DAG-induced MARCKS phosphorylation and hyperpermeability in pulmonary microvascular endothelial cells, whereas isoforms α, βI, and ϵ were dispensable with regard to these same phenomena.