Objective-To characterize matrix metalloproteinase (MMP)-2 and -9 in CSF of clinically normal dogs. Sample Population-Samples of CSF collected from 23 dogs. Procedure-Dogs were anesthetized, CSF samples were collected, and dogs were then euthanatized. Each CSF sample was evaluated immediately for RBC count, WBC count, and protein and glucose concentrations, and cytologic examination also was performed. Samples were considered normal when protein concentration was < 25 mg/dL and CSF contained < 6 WBCs/μL and < 25 RBCs/μL. Samples were stored at -70°C. Sections of brain tissue were collected and processed for histologic examination. The MMPs were evaluated by use of gelatin zymography and a polyclonal antibody-based sandwich ELISA. Results-Mean WBC count for CSF samples was < 1 WBC/μL (range, 0 to 3 WBCs/mL). Mean protein concentration was 12 mg/dL (range, 8 to 17 mg/dL). Mean RBC count was 3.65 RBCs/μL (range, 0 to 21 RBCs/μL). All CSF samples generated a clear band on zymography gels that corresponded to the human commercial standard of proenzyme MMP-2. Other major clear bands were not detected on zymography gels. Bands correlating to MMP-9 were not detected in any samples. The ELISA results revealed a mean +/- SD proenzyme MMP-2 concentration of 5.61 +/- 1.92 ng/mL (range, 3.36 to 10.83 ng/mL). Conclusions and Clinical Relevance-The proenzyme form of MMP-2 is detectable in CSF of clinically normal dogs, whereas MMP-9 is not detectable. Additional investigation of MMPs in CSF from dogs with various diseases of the nervous system is indicated.