Abstract Adoptive T cell immunotherapy can cause regression of established malignancy. One promising approach is to transfer genes encoding chimeric antigen receptors (CARs) that specifically recognize tumor-associated antigens to T cells before the T cells are adoptively transferred to patients. We have constructed a CAR that consists of an anti-CD19 single chain variable region (scFv) that is coupled to a portion of the CD28 costimulatory molecule and the signaling component of the CD3-zeta chain. CD19 is a promising target for immunotherapy because most malignant B cell express CD19, but the only normal cells that express CD19 are B cells, B cell precursors, and perhaps follicular dendritic cells. We have demonstrated that gamma-retroviruses encoding the anti-CD19 CAR can be used to transduce human T cells and that these transduced T cells specifically recognize CD19+ targets. To transduce T cells, we stimulated T cells with the anti-CD3 monoclonal antibody OKT3 on day 0 then conducted sequential retroviral transductions on day 2 and on day 3. Transductions were performed by spin-loading retroviruses onto RetroNectin (Takara) coated culture plates followed by overnight incubation of the OKT3- stimulated T cells on the plates. Forty-five to sixty-seven percent of T cells expressed the anti-CD19 CAR as measured by flow cytometry 7–8 days after transduction (n=8). Anti-CD19-CAR-transduced CD8+ and CD4+ T cells produced IFNg and IL-2 specifically in response to stimulation with CD19+ target cells. The transduced T cells specifically killed primary chronic lymphocytic leukemia (CLL) cells. T cells from CLL patients that were either untreated or previously treated with fludarabine plus rituximab could be transduced and induced to proliferate sufficiently to provide enough cells for clinical adoptive T cell transfer. In addition, we adapted this protocol for use in CLL patients with very high peripheral blood leukemia cell counts by depleting CD19+ cells using magnetic bead sorting prior to OKT3 stimulation. In preparation for a clinical trial that will enroll patients with advanced B cell malignancies, we have generated a producer cell clone that produces GALV (Gibbon ape leukemia virus)-enveloped gamma-retroviruses encoding the anti-CD19 CAR, and we have produced sufficient retroviral supernatant for the proposed clinical trial under good manufacturing practice (GMP) conditions.