The role of macrophages (M phi) in natural anti-chondrocyte cytotoxic activity of normal murine splenocytes (SPL) and peritoneal cells (PC) was examined by means of an 18-hr 51Cr-release assay. Removal of M phi by either plastic adherence or carbonyl iron and magnet resulted in significant (P less than 0.01) reduction of SPL-mediated lysis of chondrocytes. It, however, did not influence natural anti-YAC-1 leukemia activity. On the contrary, M phi-depleted suspensions of PC retained their anti-chondrocyte activity at the initial level. Neither adherent SPL nor adherent PC exerted significant anti-chondrocyte cytotoxicity. The activity of nonadherent SPL could be, however, restored by reconstitution with the graded numbers of syngeneic adherent spleen- or peritoneal cavity-derived cells. Restoration of anti-chondrocyte activity of nonadherent SPL to the level comparable with that of untreated SPL was achieved with about 10% of adherent cells. On the other hand, reconstitution of nonadherent PC did not result in an increase of chondrocyte lysis. Potentiation of nonadherent SPL activity was seen only with syngeneic M phi, and the latter could not be replaced by conditioned media which were generated in the 18-hr culture of adherent SPL or PC.