OBJECTIVE To evaluate eicosanoid concentrations in freshly prepared canine packed RBCs (PRBCs) and to assess changes in eicosanoid concentrations in PRBC units over time during storage and under transfusion conditions. DESIGN Prospective study. SAMPLE 25 plasma samples from 14 healthy Greyhounds. PROCEDURES Plasma samples were obtained during PRBC preparation (donation samples), and the PRBC units were then stored at 4°C until used for transfusion (≤ 21 days later; n = 17) or mock transfusion if expired (22 to 24 days later; 8). Immediately prior to use, 100 mL of saline (0.9% NaCl) solution was added to each unit and a pretransfusion sample was collected. A posttransfusion sample was collected after transfusion or mock transfusion. Concentrations of arachidonic acid, prostaglandin (PG) F2α, PGE2, PGD2, thromboxane B2, 6-keto-PGF1α, and leukotriene B4 were measured by liquid chromatography-mass spectrometry and analyzed statistically. RESULTS Median arachidonic acid concentration was significantly decreased in posttransfusion samples, compared with the concentration in donation samples. Median PGF2α, 6-keto-PGF1α, and leukotriene B4 concentrations were significantly increased in pretransfusion samples, compared with those in donation samples. Median PGF2α, thromboxane B2, and 6-keto-PGF1α concentrations were significantly increased in posttransfusion samples, compared with those in pretransfusion samples. Duration of PRBC storage had significant associations with pretransfusion and posttransfusion arachidonic acid and thromboxane B2 concentrations. CONCLUSIONS AND CLINICAL RELEVANCE Concentrations of several proinflammatory eicosanoids increased in PRBC units during storage, transfusion, or both. Accumulation of these products could potentially contribute to adverse transfusion reactions, and investigation of the potential association between eicosanoid concentrations in PRBCs and the incidence of transfusion reactions in dogs is warranted.