Cerebral microdialysis is a monitoring technique with expanding clinical and research utility following traumatic brain injury. This study's aim was to determine the relative recovery for 12 cytokines using both crystalloid (CNS perfusion fluid) and colloid (CNS perfusion fluid supplemented with 3.5% human serum albumin) perfusate. Six CMA71 microdialysis catheters (nominal molecular weight cut-off 100 kDa) were perfused in vitro with either crystalloid or colloid and the relative recovery (%) determined for the cytokines as follows (crystalloid/colloid perfusate): IL-1alpha (50.6/48), IL-1beta (34.6/38.4), IL-1ra (21.9/38.4), IL-2 (17.1/52.8), IL-4 (26/56.7), IL-6 (9.8/25.5), IL-8 (47.7/73.4), IL-10 (2.9/8.7), IL-17 (14.4/43.7), TNF-alpha (4.4/31.2), MIP-1alpha (31.8/55.6), and MIP-1beta (31.9/50.1). The colloid perfusate significantly improved relative recovery for nine of these cytokines ( p < 0.05), but not for IL-1alpha, IL-1beta, and IL-8. Relative recovery was related to apparent molecular weight of cytokine and to isoelectric point (pI), a surrogate marker of hydrophilicity. The mean fluid recovery for crystalloid and colloid perfusate was 92% and 145%, respectively. Scanning electron microscopy was utilized to investigate the ultrastructure of microdialysis membranes: (1) 20-kDa membrane, (2) 100-kDa membrane, and (3) ex vivo 100-kDa membrane. The 100-kDa membranes possessed multiple large cavities and the catheter examined after use in human brain clearly demonstrated cellular debris within the pores of the membrane. While colloid perfusate improves relative recovery, it causes a net influx of fluid into the microdialysis catheter, potentially dehydrating the extracellular space. This study is the first to systematically determine relative recovery in vitro for a wide range of cytokines. The two forms of perfusion fluid require direct comparison in vivo.