The aim of the study was to analyze the involvement of the endothelium in the effects of neutrophils (PMNL) on phenylephrine-pre-contracted isolated rings of the rat thoracic aorta and to compare their effects with those of peroxynitrite (ONOO−) and hypochlorous acid (HOCl). Activated PMNL-induced contraction of the precontracted aorta was prevented by the blockade of NO-synthase and by endothelium removal. In the endothelium-free preparations, the effect of PMNL reappeared in the presence of sodium nitroprusside. The effect of ONOO− and HOCl significantly differed from that of activated PMNL both in the presence and absence of the endothelium. It is therefore likely that neither ONOO− nor HOCl generated by transformation of superoxide anion radical (O2•−) produced by PMNL is involved in their action. Reduction of the relaxant effect of nitric oxide derived from the endothelium by O2•− seems to be the keystone mechanism in generation of PMNL-induced contraction.