Cynomolgus macaques were immunized by either the intramuscular (i.m.) or intranasal (i.n.) route with a HIV-1 peptide-based immunogen (C4-V3 89.6P) alone, or formulated with novel adjuvants to evaluate the ability of the adjuvants to augment peptide-specific systemic and mucosal immune responses. A mutant cholera toxin, CT-E29H, or the combination of recombinant human IL-1alpha (rhIL-1alpha) protein and recombinant human GM-CSF (rhGM-CSF) protein were tested as adjuvants for i.n. immunization, while a stable emulsion of a synthetic monophosphoryl lipid A (MPL) analogue (RC529-SE) plus rhGM-CSF protein was tested as an adjuvant for i.m. immunization. Macaques immunized i.n. with peptide alone failed to elicit an anti-C4-V3 89.6P antibody response in serum. In contrast, all the tested peptide/adjuvant formulations elicited peptide-specific immune responses. RC529-SE/rhGM-CSF elicited the highest peak anti-peptide IgG geometric mean titer in serum (1:32,768 at week 25) followed by rhIL-1alpha/rhGM-CSF (1:1217 at week 10) and CT-E29H (1:256 at week 25). Measurable SHIV neutralizing antibody responses were detectable in only one macaque immunized i.m. with peptide formulated with RC529-SE/rhGM-CSF. Macaques immunized by the i.n. route with peptide in combination with CT-E29H failed to elicit measurable antibody responses at nasal or genital mucosal surfaces. In contrast, antibody responses at the nasal and genital mucosa were detected in macaques immunized by the i.n. route with peptide in combination with rhIL-1alpha/rhGM-CSF. However, antibody responses at the nasal and genital mucosa were highest in macaques immunized parenterally with peptide in combination with the adjuvants RC529-SE/rhGM-CSF. These results suggest that parenteral vaccine administration in combination with the appropriate adjuvant formulation can elicit vaccine-specific humoral immune responses in both systemic and mucosal compartments.