MAP kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosphatase that has been shown to dephosphorylate MAP kinases. We used a "substrate-trap" technique involving a mutation in MKP-1 of the catalytically critical cysteine to a serine residue ("CS" mutant) to capture novel MKP-1 substrates. We transfected the MKP-1 (CS) mutant and control (WT) constructs into PMA-activated COS-1 cells. MKP-1-substrate complexes were immunoprecipitated, which yielded four bands of 17, 15, 14 and 10 kDa with the CS MKP-1 mutant, but not the WT MKP-1. The bands were identified by mass spectrometry as histones H3, H2B, H2A and H4, respectively. Histone H3 was phosphorylated, and purified MKP-1 dephosphorylated histone H3 (phospho-Ser 10) in vitro; whereas, histone H3 (phospho-Thr 3) was unaffected. We have previously shown that thrombin and VEGF upregulated MKP-1 in human endothelial cells (EC). We now show that both thrombin and VEGF caused dephosphorylation of histone H3 (phospho-Ser 10) and histone H3 (phospho-Thr 3) in EC with kinetics consistent with MKP-1 induction. Furthermore, MKP-1-specific (si)RNA prevented VEGF- and thrombin-induced H3 (phospho-Ser 10) dephosphorylation, but had no effect on H3 (phospho-Thr 3 or Thr 11) dephosphorylation. In summary, histone H3 is a novel substrate of MKP-1, and VEGF- and thrombin-induced H3 (phospho-Ser 10) dephosphorylation requires MKP-1. We propose that MKP-1-mediated H3 (phospho-Ser 10) dephosphorylation is a key regulatory step in EC activation by VEGF and thrombin.