Cells isolated enzymatically from the 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 the different buoyant densities of the different cells in the mixture. Macrophages (Fc gamma R+, esterase+) present in cell mixtures can by highly enriched (to 95% purity) in a single step by rosetting with opsonized erythrocytes followed by sedimentation on Lymphoprep. Separate fractions of highly purified (over 95%) macrophages obtained by the successive use of density gradients and rosetting differ significantly in the production of cytokines, e.g. cells from fractions at lower density produce little IL-6, cells from fractions at higher density are poor producers of TNF-alpha, while testicular macrophages (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.