Eicosanoids are arachidonic acid-derived mediators, with partly contradictory, incompletely elucidated actions. Thus, epoxyeicosatrienoic acids (EETs) are controversially discussed as putative vasodilatative endothelium-derived hyperpolarizing factors in the cardiovascular compartment but reported as vasoconstrictors in the lung. Inconsistent findings concerning eicosanoid physiology may be because previous methods were lacking sensitivity, identification reliability, and/or have focused on special eicosanoid groups only, ignoring the overall mediator context, and thus limiting the correlation accuracy between autacoid formation and bioactivity profile. Therefore, we developed an approach which enables the simultaneous assessment of 44 eicosanoids, including all representatives of the arachidonic acid cascade, i.e., cytochrome P450, lipoxygenase, cyclooxygenase products, and free isoprostanes as in vivo markers of oxidative stress, in one 50-minute chromatographic run. The approach combines (i) source-specific sample extraction, (ii) rugged isocratic and high-sensitivity capillary liquid-chromatographic separation, and (iii) reliable dual online photodiode-array and electrospray ionization tandem mass-spectrometric identification and quantitation. High sensitivity with limits of quantification in the femtogram range was achieved by use of capillary columns with typical high peak efficiency, due to small inner diameters, and virtually complete substance transfer to the mass spectrometer, due to flow rates in the low microliter range, instead of large inner diameter columns with low chromatographic signal and only partial analyte transfer employed by previous methods. This expeditious, global and sensitive technique provides the prerequisite for new, accurate insights regarding the physiology of specific mediators, for example EETs, in the context of all relevant vasoactive autacoids under varying conditions of oxidative stress by direct comparison of all eicosanoid generation profiles. Indeed, application of comprehensive "eicoprofiling" to hypoxically ventilated rabbit lungs revealed at a glance the enhanced biosynthesis of free EETs in the overall mediator generation context, thus suggesting their hypothetical contribution to hypoxic pulmonary vasoconstriction.