Cells isolated enzymatically from interstitial tissue of mouse male gonads are composed of macrophages, Leydig cells, and myofibroblasts. They can be separated on density gradients either by sedimentation (Ficoll) or flotation (Percoll) into several fractions according to different buoyant density containing mixtures of different cells. Macrophages (FcR+, esterase+) present in cell mixtures can by highly enriched in a single step to 95% purity by rosetting with opsonized erythrocytes followed by sedimentation on Lymphoprep. Separate fractions of highly purified (over 95%) macrophages obtained by successive use of density gradients and rosetting differ significantly in the production of cytokines, such as cells from fractions at lower density produce little IL-6, cells from fractions at higher density are poor producers of TNF-alpha whereas TMf in intermediate fractions produce significant amounts of both cytokines. These differences may suggest that particular subpopulations of testicular macrophages play different biological roles in the testis.