The rete testis and subcapsular regions of the testis are sites of disease onset in murine experimental auto-immune orchitis, but macrophages in these compartments are not well-characterised. Macrophages were localized by immunohistochemistry using an anti-F4/80 antibody in testis sections from adult wild-type mice (n=10-17) and mice with a GFP-expressing transgene at the locus of the macrophage receptor, CX3CR1 (Cx3cr1gfp/+;n=6). Sections were also co-labelled by immunofluorescence for the anti-inflammatory marker, CD206, and the antigen-presenting MHC class II molecule (I-A/1-E) to identify activated macrophages. Sections were scanned (Olympus VS120 slide scanner), and macrophages were enumerated by stereology. Compared with the parenchyma surrounding the seminiferous tubules, the volume density of interstitial macrophages (30 macrophages/µm3) was 10-fold higher in the rete testis, and the density of peritubular macrophages (3.5 macrophages/µm3) was 3-fold higher. Macrophage density between the tunica albuginea and tubules (subcapsular region) was similar to the rest of the parenchyma, and macrophages were observed within the tunica albuginea itself. In contrast to interstitial macrophages between the seminiferous tubules, which have low MHCII expression, most interstitial macrophages in the rete testis and subcapsular region were CX3CR1+F4/80+MHCIIhigh. Peritubular macrophages in the rete testis were CX3CR1+F4/80+MHCIIhigh, but also expressed CD206, unlike peritubular macrophages of the seminiferous tubules. These data indicate that the majority of macrophages within the rete testis and subcapsular regions differ from macrophages in the rest of the parenchyma in that they have an activated, but possibly anti-inflammatory, phenotype. Accumulation of these macrophages and the phenotype of the peritubular macrophages within the rete testis suggest that they may play a role in recognizing sperm antigens and inducing tolerance to mature sperm leaving the testis, in addition to providing protection against ascending infections of the genital tract. We also predict that alterations in the function of these macrophages during inflammatory disease may cause sperm autoimmunity.