The aim of this study was to evaluate the effect of an ultrapure bovine stroma-free hemoglobin (SFH) on pulmonary vascular resistance and mediator release and to analyze potential mechanisms of action in the isolated perfused rabbit lung model. Repetitive bolus applications of small amounts of bovine SFH were examined which resulted in a reproducible acute increase of pulmonary vascular resistance of approx. 9 mmHg (controls, n = 6). It was tested whether the platelet-activating factor (PAF) antagonist WEB 2086 (50 μM; n = 6), the cyclooxygenase blocker diclofenac (10 μg/ml; n = 6), the iron-chelating agent deferoxamine (500 μg/ml, n = 6) and the radical scavenger catalase (5000 U/ml; n = 6) exert a protective effect on vasoconstrictor response to SFH. The pressure increase was completely suppressed in the lungs pretreated with WEB 2086, whereas diclofenac, deferoxamine and catalase failed to inhibit the vasoconstriction due to SFH. No significant differences in either TXB2 generation or in histamine release were found in the WEB 2086 group compared with untreated lungs. The results point towards the crucial role of PAF in mediation of vasoconstrictor side effects due to SFH.