Objective. To examine the role of dipeptidyl peptidase I (DPPI), a widely expressed lysosomal cysteine protease, in the development of collagen-induced arthritis (CIA) in mice. Methods. Wild-type (WT) and DPPI-deficient (DPPI -/-) mice backcrossed to DBA/1J mice for 10 generations were immunized with bovine type II collagen (CII), and disease susceptibility and severity were assessed over time. Collagen-specific B cell and T cell responses and the production of proinflammatory cytokines (tumor necrosis factor α, interleukin-1, and interleukin-6) were measured. In addition, adoptive transfer of splenocytes from WT, CII-sensitized mice was performed to evaluate the specific role of DPPI-/- T lymphocytes. Results. The majority of DPPI-/- mice were resistant to CIA induction, although clinical disease (i.e., evidence of inflammation and bone erosions) did develop in a small number of DPPI-/- mice. The protection against disease development was not attributable to a defect in the B and T cell response to collagen immunization, because both anticollagen antibody production and T cell proliferation in response to CII were normal. Release of the proinflammatory cytokines was largely unaffected in CII-stimulated DPPI-/- splenocytes. In addition, when cells isolated from the joints of DPPI -/- mice were stimulated in vitro, they had no intrinsic defect in their ability to release inflammatory cytokines. Last, adoptive transfer of splenocytes from WT, CII-immunized mice into naive WT and DPPI-/- mice led to development of arthritis in WT mice but not in DPPI-/- mice. Conclusion. These results indicate that DPPI regulates a critical step in the development of CIA that is independent of T cell and B cell functions.