Although microglia isolation from embryonic or postnatal mouse brain is possible using a number of different protocols, microglia isolation from adult brain is more challenging and often results in low yields. Here, we describe a protocol to isolate intact microglia from adult mouse brain for functional assays, immunocytochemistry, and/or flow cytometry analysis. This protocol involves enzymatic dissociation in medium supplemented with dispase II, papain, and DNase I followed by mechanical dissociation. Cell separation is achieved via percoll gradients of various densities. Microglia isolated using this protocol is suitable for flow cytometry analysis, RNA isolation for gene expression by real-time PCR or microarrays, and for functional assays including cytokine production, chemotaxis, and phagocytosis.