Immunohistochemistry is widely used to identify immune cells, but reliable detection of antigens is not always compatible with good-quality fixation. This is particularly problematic for rodent testes, where preservation of morphology is crucial. Testicular cells can be differentiated in well-fixed, paraffin-embedded sections, but morphometric analysis using morphological criteria is time-consuming. Furthermore, testicular interstitial macrophages may be easily discriminated, but the peritubular macrophages are difficult to visualise. The aim of this study was to optimise and validate methods for detection and quantification of macrophages using several histological stains and immunohistochemical protocols in Bouin’s-fixed, paraffin-embedded testes from mice with altered levels of the immunoregulatory cytokine, activin and its binding protein, follistatin: (a) Inhba+/- (reduced activin), (b) Inha-/- (activin over-production), and c) TghFST315 transgenics (reduced follistatin). May-Grunwald-Giemsa stain provided the best discrimination between macrophages and other interstitial cells, based on differences in nuclear morphology and chromatin patterns, but identification of peritubular macrophages was less reliable using this approach. Optimisation of antigen retrieval, serum blocking, washing and primary to secondary antibody concentrations for F4/80 immunohistochemistry to detect macrophages was established. Peritubular macrophages were readily discriminated using this optimised method and there was a good correlation between the numbers of interstitial macrophages identified by histology and immunohistochemistry. However, preliminary morphometric analysis indicated no significant differences in total macrophage numbers between the various activin/follistatin mouse models. This was also verified by F4/80 and CD45 gene expression measured by qRT-PCR. These studies represent a significant methodological advancement in the discrimination and quantification of macrophages in well-preserved mouse testis tissue. These preliminary results suggest that alterations in endogenous activin or follistatin have minimal effects on the total number of peritubular and interstitial macrophages in the adult mouse testis. Optimisation of these techniques to quantify macrophage functional subsets and other immune cells in the testis is in progress.