In an effort to develop a safe Nef component for use in Cytotoxic T-lymphocyte (CTL)-based HIV-1 vaccines, several versions of Nef constructs lacking myristoylation and dileucine motif were engineered and their abilities to elicit T cell responses were evaluated in mice. Nef-specific murine T cell epitopes were first mapped in three strains of mice (Balb/c, C3H/HeN and C57BL/6), and a pair of dominant Nef-specific CD4(+) and CD8(+) T cell epitopes were identified in C57BL/6 mice. C57BL/6 mice were subsequently immunized with engineered Nef DNA constructs, and Nef-specific CD4(+) and CD8(+) T cell responses were determined. A Nef mutant with simple alanine substitutions at the myristoylation and dileucine sites was impaired in its ability to elicit Nef-specific CD4(+) and CD8(+) T cell responses. Addition of human tissue plasminogen activator (TPA) leader sequence to the N terminus of Nef, which concomitantly inactivates the myristoylation site, significantly enhanced the Nef-specific T cell responses. These findings may have practical implications for developing HIV-1 Nef vaccine component.