If hyperacute rejection is prevented in the guinea-pig (GP)-to-Lewis rat (Lew) cardiac xenograft (CXg) model, an accelerated rejection involving cellular infiltration occurs in 3 to 4 days. In previous work using an adoptive transfer model, we found that this accelerated rejection was facilitated by either sensitized splenocytes or sensitized serum. In the current study, in an attempt to determine which splenocyte subset(s) facilitated this process, sensitized splenocytes, with or without subset depletion were injected, into complement- and natural antibody-depleted Lew recipients of GP CXgs. Graft survival was 4.18±0.75 days with no injection (n=ll), 4.13±0.99 days with naive splenocytes (n=8), 1.80±0.45 days with sensitized splenocytes (n=5), 2.67±1.03 days with CD4(W3/25+) depletion of the sensitized splenocytes (n=6), 3.13±0.84 days with CD8(OX8+) cell depletion (n=8), 4.70±0.68 days with macrophage depletion (n=10), and 4.22±0.41 days with В cell depletion (n=9). Cellular infiltrates, hemorrhage, myocyte necrosis, and endothelial deposition of IgG, IgM, and fibrin were seen in rejected grafts. In most groups, infiltrating cells consisted of CD4 (W3/25+), CD8 (OX8+), IL2R+cells, macrophages, and natural killer (NK) cells. However, in the macrophages-depleted group, activated (ED2+) macrophages and NK cells were significantly reduced. Total IgM, anti-GP IgM, and anti-GP IgG rebounded in all groups over several days but were not consistent at the time of rejection. Lewis rats rejecting GP CXgs early had lower final titers than those rejecting later. Total IgG titers rebounded to baseline by posttrans-plant day 1 and were therefore similar in all groups at the time of rejection. These findings suggest that this accelerated rejection requires interaction between macrophages and В cells, since depletion of either signifícantly alters the rejection tempo. A possible explanation is that xenoreactive IgG antibodies, synthesized by sensitized В cells, bind their target antigens— but also bind sensitized macrophages through their Fc region, thus causing rejection by antibody-dependent cell-mediated cytotoxicity.