Popliteal lymph nodes (LN) of mice were depleted of their macrophage (M phi) populations in the subcapsular sinus and medulla by subcutaneous injection of dichloromethylene diphosphonate (Cl2MDP)-containing liposomes into the footpads. Complete restoration of both M phi populations could be observed as late as 5 months after liposome administration. This relatively long repopulation time could be due to a depot of liposomes, directly killing all M phi precursors after extravasation into the interstitial tissue of the footpad. On the other hand, local interstitial precursors with very low turnover rates may have been depleted in the interstitial tissue of the hind leg. Therefore, two different types of experiments were performed; one in which M phi-depleted LN were replaced by control LN at various time points after liposome treatment, and another whereby M phi-depleted LN were transplanted into control animals. When liposome-treated, M phi-depleted LN were transplanted into control animals, a complete restoration of both populations in the subcapsular sinus and medulla could be observed within 5 weeks. Control LN transplanted into a Cl2MDP-liposome-treated leg showed a rapid disappearance of M phi from the subcapsular sinus and medulla and these cell populations remained absent for at least 7-8 weeks after liposome treatment, when the first cells started to reappear. Complete repopulation of these areas by M phi took as long as 15 weeks. Using labeled liposomes the presence of a continuous liposome depot was found to be very unlikely. These results suggest that the population of precursor cells that will give rise to M phi in the subcapsular sinus and medulla of a LN is probably contained within the interstitial tissue and is almost independent of precursor supply from the blood compartment.