In this study, we investigated the possibility of selective depletion of donor alloantigen-specific T cells from C57BL/6 (H-2^b) mice to prevent graft-versus-host disease (GVHD). These cells were first activated with irradiated BALB/c (H-2^d) host spleen cells in a 5-day mixed lymphocyte culture. Following this activation, a photoactive rhodamine derivative called 4,5-dibromorhodamine 123 (TH9402), was added. This compound is selectively retained in the mitochondria of activated host-reactive cells but not tumor- or third-party–specific resting cells. The treated cells were subsequently exposed to visible light (514 nm) to deplete the TH9402-enriched activated host-reactive cells. Treatment with photodynamic cell purging process (PDP) inhibited antihost responses measured by cytotoxic T lymphocytes (CTL) by 93%, and interferon-γ production by 66%. By contrast, anti-BCL1 (BALB/c-origin leukemia/lymphoma) and anti–third-party C3H/HeJ (H-2^k) responses were preserved. PDP-treated primed C57BL/6 cells were further tested in vivo. All lethally irradiated BALB/c mice inoculated with BCL1 cells and T-cell–depleted bone marrow cells developed leukemia by day +30, with 50% mortality by 100 days. All mice died of GVHD after addition of 5 × 10⁶untreated primed C57BL/6 cells. However, addition of same numbers of PDP-treated cells allowed 90% of the recipients to survive more than 100 days without detectable BCL1 tumor cells and free of GVHD. Moreover, PDP-treated primed C57BL/6 cells retained the ability to induce GVHD in the third-party C3H/HeJ mice. These data suggest that PDP can selectively deplete host alloantigen-specific T cells for GVHD prevention and immune and antileukemia function preserve.