Overexpression and activation of protein kinase C-ε (PKCε) results in myocardial hypertrophy. However, these observations do not establish that PKCε is required for the development of myocardial hypertrophy. Thus, we subjected PKCε-knockout (KO) mice to a hypertrophic stimulus by transverse aortic constriction (TAC). KO mice show normal cardiac morphology and function. TAC caused similar cardiac hypertrophy in KO and wild-type (WT) mice. However, KO mice developed more interstitial fibrosis and showed enhanced expression of collagen Iα1 and collagen III after TAC associated with diastolic dysfunction, as assessed by tissue Doppler echocardiography (Ea/Aa after TAC: WT 2.1±0.3 versus KO 1.0±0.2; P<0.05). To explore underlying mechanisms, we analyzed the left ventricular (LV) expression pattern of additional PKC isoforms (ie, PKCα, PKCβ, and PKCδ). After TAC, expression and activation of PKCδ protein was increased in KO LVs. Moreover, KO LVs displayed enhanced activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), whereas p42/p44–MAPK activation was attenuated. Under stretch, cultured KO fibroblasts showed a 2-fold increased collagen Iα1 (col Iα1) expression, which was prevented by PKCδ inhibitor rottlerin or by p38 MAPK inhibitor SB 203580. In conclusion, PKCε is not required for the development of a pressure overload–induced myocardial hypertrophy. Lack of PKCε results in upregulation of PKCδ and promotes activation of p38 MAPK and JNK, which appears to compensate for cardiac hypertrophy, but in turn, is associated with increased collagen deposition and impaired diastolic function.