ABSTRACT Dendritic cells (DCs), which are potent antigen-presenting cells (APCs), are used as adjuvants for the treatment of cancer and infectious diseases in human and nonhuman primates, with documented clinical efficacy. The hepatitis C virus (HCV)-chimpanzee model is the best available model for testing the immunotherapeutic effects of DCs in the setting of a chronic infection, as chimpanzees develop a persistent infection resembling that seen in humans. However, several reports have suggested that DCs derived from chronically infected individuals or nonhuman primates are functionally compromised. As a prelude to clinical studies, we evaluated whether functionally mature DCs could be generated in chimpanzee plasma by good manufacturing practice using CD14 ⁺ mononuclear precursors from chronically infected chimpanzees. DCs generated in a medium with HCV-negative plasma and treated with a defined cocktail of cytokines or a CD40 ligand trimer matured fully, as measured by the induction of CD83 expression and the upregulation of costimulatory molecules. Furthermore, the expression of CCR7 was induced, suggesting an acquisition of migration capacity. Mature DCs were capable of stimulating allogeneic T cells, antigen-specific memory CD4 ⁺ T cells, and HCV-specific CD8 ⁺ -T-cell clones. In all cases, there was no evidence of HCV infection in DCs. Furthermore, these DCs maintained their phenotype and APC function after cryopreservation. Finally, no discernible differences were noted between DCs derived from HCV-infected and uninfected chimpanzees. In summary, precursor cells from HCV-infected chimpanzees are fully capable of differentiating into functional, mature DCs, which can now be reproducibly prepared for investigations of their immunotherapeutic potential in the setting of chronic HCV infection.