The partition of cis-parinaric acid (9,11,13,15-cis, trans, trans,cis-octadecatetraenoic acid, cis-PnA) and trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid, trans-PnA) among aqueous, solid lipid, and fluid lipid phases has been measured by three spectroscopic parameters: absorption spectral shifts, fluorescence quantum yield, and fluorescence polarization. The solid lipid was dipalmitoylphosphatidylcholine (DPPC); the fluid lipid was palmitoyldocosahexaenoylphosphatidylcholine (PDPC). Mole fraction partition coefficients between lipid and water were determined by absorption spectroscopy to be for ci--PnA, 5.3 X 10(5) with a solid lipid and 9 X 10(5) with fluid lipid and, for trans-PnA, 5 X 10(6) with solid lipid and 1.7 X 10(6) with fluid lipid. Ratios of the solid to the fluid partition coefficients (Kps/f) are 0.6 +/- 0.2 for cis-PnA and 3 +/- 1 for trans-PnA. A phase diagram for codispersions of DPPC and PDPC has been constructed from the measurements of the temperature dependence of the fluorescence quantum yield and polarization of cis-PnA and trans-PnA and their methyl ester derivatives. A simple analysis based on the phase diagram and fluorescence data allows additional calculations of Kps/f's which are determined to be 0.7 +/- 0.2 for the cis probes and 4 +/- 1 for the trans probes. The relative preference of trans-PnA for solid phase lipids and its enhanced quantum yield in solid phase lipids make it sensitive to a few percent solid. The trans probes provide evidence that structural order may persist in dispersions of these phospholipids 10 degrees C or more above their transition temperature. It is concluded that measurements of PnA fluorescence polarization vs. temperature are better suited than measurements of quantum yield vs. temperature for determining phospholipid phase separation.