AbstractMicrodialysis is an established sampling technique for the in vivo measurement of a variety of substances in both blood and tissue. These include neurotransmitters and neuropeptides, enzymes, and electrolytes, as well as various hormones and pharmaceutical agents. More recently, microdialysis has been used to infuse exogenous as well as endogenous substances into the brain and spinal cord. In microdialysis, a semipermeable dialysis membrane is introduced into the fluid or tissue compartment to be sampled and perfused with physiological fluid. As a result of passive diffusion, molecules migrate across the membrane along their concentration gradient. Molecules found in high concentrations within the tissue compartment migrate across the membrane into the dialysis tubing where they can be collected for subsequent quantification, whereas molecules found in high concentrations within the membrane diffuse outward into the surrounding tissue compartment. This unit describes construction of concentric and side‐by‐side microdialysis probes, which differ with respect to size and inflow/outflow tube compositions, as well as a modification of a horizontal probe. This unit also covers methods for surgically implanting microdialysis probes in rats and mice and for conducting microdialysis in vitro and in vivo in rodents. Finally, procedures are provided for carrying out quantitative microdialysis techniques.