BACKGROUND: In comparison to the classical isoforms of protein kinase C (PKC), the novel isoforms are thought to play minor or inhibitory roles in the regulation of platelet activation and thrombosis. OBJECTIVES: To measure the levels of PKCθ and PKCε and to investigate the phenotype of mice deficient in both novel PKC isoforms. METHODS: Tail bleeding and platelet activation assays were monitored in mice and platelets from mice deficient in both PKCθ and PKCε. RESULTS: PKCε plays a minor role in supporting aggregation and secretion following stimulation of the collagen receptor GPVI in mouse platelets but has no apparent role in spreading on fibrinogen. PKCθ, in contrast, plays a minor role in supporting adhesion and filopodial generation on fibrinogen but has no apparent role in aggregation and secretion induced by GPVI despite being expressed at over 10 times the level of PKCε. Platelets deficient in both novel isoforms have a similar pattern of aggregation downstream of GPVI and spreading on fibrinogen as the single null mutants. Strikingly, a marked reduction in aggregation on collagen under arteriolar shear conditions is observed in blood from the double but not single-deficient mice along with a significant increase in tail bleeding. CONCLUSIONS: These results reveal a greater than additive role for PKCθ and PKCε in supporting platelet activation under shear conditions and demonstrate that, in combination, the two novel PKCs support platelet activation.