Transient hypoxia stimulates proliferation of endothelial cells via endogenous signalling mechanisms, including generation of ROS by mitochondria and NADPH oxidase as well as activation of the MEK/ERK pathway. Here it was analysed whether p38 MAPK is a downstream signalling element of the hypoxia-induced proliferation. Exposure of porcine aortic endothelial cells to hypoxia (2h, Po2 < 10 mmHg) caused an increase in phosphorylation of retinoblastoma protein (pRb~P, Western blot), DNA synthesis (BrdU incorporation) and increase in cell number after 24h of reoxygenation (Po2 = 140 mmHg). Inhibition of MEK by PD98059 attenuated hypoxia-induced activation of NADPH oxidase and p38 MAPK, indicating that both signalling elements are downstream of MEK/ERK. Inhibition of NADPH oxidase by antisense oligonucleotides (AS) against NADPH oxidase-subunit p22phox reduced p38 MAPK activation and abolished the hypoxic proliferation response, indicating that NADPH oxidase is upstream of p38 MAPK. Immunoprecipitation of p38 MAPK showed, that hypoxia stimulated phosphorylation of a and [gamma] isoforms of p38 MAPK, but neither b nor [delta]. Inhibition of p38 MAPKa by AS p38 MAPKa abolished pRb~P and cell proliferation. Conclusion: These results indicate that in endothelial cells p38 MAPKa is a central signalling element, downstream of MEK/ERK and NADPH oxidase in the proliferation response to transient hypoxia.