Autolymphocyte therapy (ALT) is adoptive cellular therapy of neoplastic disease using ex vivo activation of autologous (human) or syngeneic (murine) lymphocytes from tumor-bearing hosts (TBH) by low doses of anti-CD3 monoclonal antibody (MAb) and a mixture of previously prepared autologous cytokines (T3CS). Ex vivo activation by T3CS without tumor antigen results in expansion of CD44+ (memory) T cells. These memory T cells (ALT cells) mediate in vivo anti-tumor specificity and with cyclophosphamide (CY) are capable of curing metastatic disease in murine TBH. To determine whether CY could enhance the effectiveness of CD4+ or CD8+ subsets of ALT cells, C57BL/6) TBH with B16 melanoma or Lewis lung (3LL) carcinoma were treated with adoptive chemoimmunotherapy (ACIT) using CD4-depleted or CD8-depleted ALT cells and CY. ALT cells were derived from splenocytes of B16 or 3LL-TBH and activated ex vivo with T3CS. Depletion of CD4+ or CD8+ T cells was performed before or after activation with T3CS. B 16-TBH or 3LL-TBH that received AClT using CY with B 16-derived or 3LL-derived CD8-depleted ALT cells, respectively, demonstrated cure of metastatic disease regardless of whether CD8+ T cells were depleted before or after T3CS activation. B 16 or 3LL-TBH that received AClT using CY with 816 or 3LL-derived CD4-depleted ALT cells also cured metastatic disease but only if CD4+ T cells were depleted after T3CS activation. lnterleukin (1L)-2 added to pre-T3CS CD4-depleted ALT cells cultured with T3CS restored anti-tumor activity when combined with CY. TBH cured by AClT using CY and ALT-cell subsets derived from syngeneic TBH with the identical tumor displayed tumorspecific immunity in rejecting a lethal challenge of identical but not reciprocal tumor. TBH given AClT using CY and ALT-cell subsets derived from splenocytes of syngeneic TBH with reciprocal tumors rejected lethal challenges of both tumors. Tumor specificity measured by interferon (IFN)-y and %r-release assays was demonstrated in pre-or post-T3CS/CD8-depleted, post-T3CS/CD4-depleted and pre-T3CS + IL-Z/CD4-depleted ALT-cell subsets. Our data demonstrate that AClT using CY combined with ex vivo T3CS-activated CD44+ memory T-cell subsets conveys long-term tumor-specific immunity.