The role of the spleen in the induction and maintenance of unresponsiveness to skin allografts and in the generation of suppressor cells has been studied in ALS-treated B6AF1 mice grafted with C3H/He skin and injected with C3H/He marrow. B6AF1 mice were splenectomized either before the induction of unresponsiveness or on day +13, +28, or +42 after unresponsiveness was induced. Graft survival in the splenectomized mice was compared to that observed in nonsplenectomized ALS-treated, marrow-injected controls. Graft survival was prolonged equally in all splenectomized groups and the nonsplenectomized controls. To study the effect of the spleen on the generation of suppressor cells, lymph node cells were removed at day +42 from splenectomized ALS-treated, marrow-injected B6AF1 mice bearing C3H/He skin grafts and transferred to ALS-treated B6AF1 recipientso ALS-treated BTAF1 recipients grafted with C3H/He skin. Graft survival in the secondary recipients receiving lymph node cells from splenectomized donors was compared to that observed in ALS-treated B6AF1 mice that received lymph node cells transferred from nonsplenectomized enhanced donors. Suppressor cell activity could be detected in the nodes of splenectomized mice, but a higher dose of lymph node cells was required to transfer unresponsiveness from splenectomized donors compared to nonsplenectomized donors. These results indicate that the spleen is not necessary for the induction or maintenance of unresponsiveness to skin allografts in ALS-treated, marrow-injected mice. In addition, suppressor cells can be generated in the lymph nodes of unresponsive mice in the absence of the spleen, although the production of suppressor cells appears to be less effective in splenectomized mice than in mice with intact spleens.