Peptides based on the pseudosubstrate (PS) sequence of conventional protein kinase C isoenzymes (alpha, beta, gamma) specifically inhibit PKC activity in permeabilized cells, but whether PS can be used to study the role of PBC in the proliferation or migration of intact endothelial cells (EC) and angiogenesis is unknown. Peptides based on the PKC eta pseudosubstrate (eta PS) sequence were 3.5- to 8-fold more potent in inhibiting the PKC alpha, delta, epsilon, or eta kinase activity than was the peptide based on the PKC alpha pseudosubstrate (alpha PS) sequence. Thus, eta PS was conditionally overexpressed in intact EC and compared to alpha PS, Serum-induced growth of EC expressing eta PS was significantly slower than that of control EC. eta PS EC demonstrated slower rate of serum stimulated migration than that of either control or (UPS EC. Expression of either eta PS or alpha PS produced slower rates of PMA induced EC migration, as compared to control EC. In an in vitro three-dimensional assay in which EC organize into capillary tubules, the EC that expressed eta PS formed fewer such tubules. This study shows that pseudosubstrate inhibitors derived from PKC eta are more potent both in vitro and in vivo than one based on the conventional isoenzyme PKC alpha. These data further support a role for PKC in proliferation and migration of intact EC, and angiogenesis.