The aim of this study was to determine the regulation by MKP-1 of MAPK activity and protein expression in cardiomyocyte hypertrophic response induced by Ang II. Neonatal rat cardiomyocyte hypertrophic response was assayed by cell surface area, protein synthesis rate and protein content. MAPK activity was determined by an in-gel kinase assay. Protein expression of MAPK and MKP-1 were detected by Western blotting. The results are as follows. (1) Ang II induced promotion of (3)H-leucine incorporation and increase in cell protein content and cell surface area in a dose-dependent manner. Pretreatment with a selective AT(1) receptor antagonist CV11974 or a specific MEK inhibitor PD098059, cardiomyocyte hypertrophic response induced by Ang II was inhibited by 85% and 32.5%, respectively. (2) After pretreatment with PD098059 or CV11974, AngII-induced increases in p44MAPK and p42MAPK protein expression and enzyme activity (expressed by gamma-(32)P-ATP incorporation) were all inhibited obviously. (3) With treatment of myocytes by Ang II for 5 min, MAPK activity determined by p44MAPK and p42MAPK protein expression began to increase, while MKP-1 protein expression was detected within 30 min and lasted more than 2 h following treatment with Ang II. (4) Pretreatment of cardiomyocytes with actinomycin D (3 microgram/ml) for 30 min inhibited MKP-1 protein expression, while p44MAPK and p42MAPK protein expression was still detected 120 min after Ang II treatment. The above results demonstrate that activation of MAPK plays an important role in Ang II-induced cardiomyocyte hypertrophic response in neonatal rat cardiomyocytes through MKP-1 mediated inactivation of p44MAPK and p42MAPK.cardiomyocyte hypertrophic response in neonatal rat cardiomyocytes through MKP-1 mediated inactivation of p44MAPK and p42MAPK.