Microdialysis emerges as a useful tool to evaluate tissue inflammation in a number of clinical conditions, like sepsis and transplant rejection, but systematic methodological studies are missing. This study was undertaken to determine the recovery of relevant inflammatory mediators using the microdialysis system, comparing microdialysis membranes with two different molecular weight cut-offs at different flow rates. Twenty and 100 kDa pore sizes CMA microdialysis catheters were investigated using velocities of 0.3, 1.0 and 5.0 μl/min. Reference preparations for cytokines [tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10; m.w. 17–28 kDa] and chemokines (IL-8, MCP-1, IP-10 and MIG; m.w. 7–11 kDa) were prepared from plasma after incubating human whole blood with lipopolysaccharide. Reference preparation for complement anaphylatoxins (C3a, C4a, C5a; m.w. 9–11 kDa) was prepared by incubating human plasma with heat-aggregated immunoglobulin G. The reference preparations were quantified for the respective inflammatory molecules and used as medium for the microdialysis procedure. Through the 20 kDa filter only the four chemokines passed, but with low recovery (3–7%) and limited to the 1.0 μl/min velocity. The recovery with the 100 kDa filter was as follows: IL-1β = 75%, MCP-1 = 55%, MIG = 50%, IL-8 = 38%, C4a = 28%, IP-10 = 22%, C5a = 20%, C3a = 16%, IL-6 = 11, IL-10 = 8% and TNF-α = 4%. The highest recovery for all chemokines and anaphylatoxins were consistently at velocity 1.0 μl/min, whereas IL-1β and IL-10 recovered most efficiently at 0.3 μl/min. Thus, microdialysis using catheters with a cut-off of 100 kDa is a reliable method to detect inflammation as judged by a defined panel of inflammatory markers. These findings may have important implications for future clinical studies.