MMP-9 (gelatinase B) is recognized in chronic obstructive pulmonary disease (COPD) and now asthma as playing a central role in matrix degradation in injury, as well as contributing to the remodeling process. The increasing focus on MMP-9 in human and animal research supports the need for a reliable immunostain in lung tissue. However, MMP-9 immunostaining in murine systems is hampered by several factors. First, many of the anti-human antibodies do not readily cross-react with murine MMP-9 despite the high degree of conservation between human and murine MMP-9. Secondly, the availability of detailed protocols is limited. Lung MMP-9 immunostaining is further complicated by technical issues such as edge effect, availability of positive and negative controls, antigen retrieval, staining specificity, and the need to achieve a delicate balance of primary and secondary antibody concentrations, and colorimetric reagents which will allow visualization of specific cell expression in highly delicate lung tissue, while also demonstrating adequate uptake in (extra-pulmonary) tissue controls. We describe a detailed method for immunostaining MMP-9 in mouse lung paraffin-embedded tissue utilizing human ovary as a control since MMP-9 is known to be over-expressed in human ovarian carcinomas.