Microdialysis (mD) is the predominant technique for measurements of brain chemistry, especially when such measurements are made in awake animals. Relatively little attention has been paid, however, to the potential effect on such measurements of variation in the perfusate solution used. Here, we report that small variations in the ionic composition of microdialysis perfusate produced four-fold differences (0.53-2.18 mM) in the results obtained when measuring brain extracellular fluid (ECF) glucose. These changes may be linked to concomitant alterations of local neural activity caused by the perfusate composition. In addition to perfusate composition, probe type also proved to have a significant impact on microdialysis measurements. Further, we report the first direct microdialysis measurements of brain ECF ionic composition, showing significant differences from that of the cerebro-spinal fluid (CSF). Modifying the ionic composition of microdialysis perfusate based on these measurements resulted in a measured hippocampal ECF glucose level of 1.26 +/- .04 mM. Increased understanding of the impact of differences in the perfusate solutions used by different laboratories may provide a basis for reconciliation of apparently disparate microdialysis results within the literature.