Analysis of fresh plasma from normal volunteers by negative ion chemical ionization GC/MS reveals what appear to be multiple PGF2 compounds with levels ranging from approximately 5 to 40 pg/ml. Interestingly, storage of plasma at -20 degrees C for several months was found to markedly increase the levels of these compounds to about 1000-4000 pg/ml, approximately 50-fold higher than levels detected in fresh plasma. Further studies aimed at understanding this observation revealed that alkaline hydrolysis of plasma lipids also yielded quantities of these compounds in the range that were detected in stored plasma. Employing a number of approaches such as deuteriated derivatives, hydrogenation, immunoreactivity with an anti-9 alpha, 11 beta-PGF2 antibody, and electron ionization mass spectral analysis, convincing evidence was obtained that these compounds in both stored and base-treated plasma were in fact PGF2 compounds. Formation of these compounds was found to occur by a nonenzymatic oxidative process in that the antioxidant, butylated hydroxytoluene, and the reducing agent, triphenylphosphine, markedly suppressed their formation. Evidence is presented to support a proposed mechanism that oxidative formation of these compounds involves the formation of endoperoxide intermediates which are directly reduced by naturally occurring biological substances to PGF2 compounds. Formation of these compounds occurs very readily in biological fluids. This finding has important ramifications not only for analysis of enzymatically derived PGF2 compounds but also for other eicosanoids which can be formed by this same nonenzymatic process. These analytical concerns apply to both immunoassay methods and physical methods of analysis such as gas chromatography/mass spectrometry.