Cytolytic T cells (CTL) play a critical role in providing protection against the liver stage of malaria infection. Previous investigations have shown that induction of CTL against peptide or proteins can be achieved by attachment of lipids. In the present study, we used the Plasmodium berghei circumsporozoite protein CTL epitope (SYIPSAEKI (PL76)). This peptide with cysteine-serine (CS) as spacer amino acids was coupled to palmitic acid (PA). The same CTL epitope containing only an extra serine was linked to S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-cysteine (tripam-C). Inbred mice [(BALB/c x C57BL/6)F1] were immunized intravenously with the lipopeptides. Both types of lipopeptides induced significant CTL responses after one injection. Immunization of the monopalmitic acid-peptide conjugate intraperitoneally emulsified in Freund's complete adjuvant also induced a significant CTL response, but the magnitude was lower as compared to the intravenous route. The major advantages of the use of the simple monopalmitic acid-peptide conjugates are: (i) low costs of the fatty acid; (ii) coupling of lipid to peptide can be performed using the peptide synthesizer during standard peptide synthesis, and (iii) standard peptide methodology can be used for purification. To investigate whether a spacer amino acid sequence between the actual CTL epitope and PA is required for induction of an optimal CTL response, we prepared monopalmitic acid-peptide conjugates with different spacer amino acids. A lipopeptide without a spacer amino acid and another one containing the CS spacer sequence both induced a CTL response, whereas a lipopeptide with a serine as spacer failed to induce CTL. These results indicate that the amino acid spacer sequences influence the immunological properties of the palmitic acid-peptide conjugates.