The immunologically dominant central portion of the circumsporozoite (CS) surface protein on the malaria parasite Plasmodium falciparum contains a large number of tandemly repeated NPNA tetrapeptide motifs. The preferred secondary structure of this repeat unit in aqueous solution has been investigated with the aid of the secondary structure-inducing amino acid (8-a-methylproline (PMe). 'H-Nuclear magnetic resonance (Nh4R) and circular dichroism (CD) spectroscopy have been used to probe the structures of synthetic peptides containing one to three tetrapeptide NPMeNA units. The far-UV CD spectra of these peptides show more intense negative bands at 215 nm than do similar peptides based on the NPNA motif. This and the temperature dependence of the peptide amide chemical shifts, the pattern of NOE connectivities, and the magnitude of 3J coupling constants, derived from oneand two-dimensional NMR spectra of Ac(NPMeNA)3-OH, provide strong evidence for stable tumlike structures. From NOE distance and dihedral angle restraints, structures consistent with the NMR parameters were calculated. These reveal a stable hydrogen-bonded type-I p-tum conformation (most likely present at 70-80% population) within each NPMeNA motif, stabilized by the backbone C, methylation. Side chain to backbone hydrogen bonds involving the side chain amide groups of both asparagine residues also appear to impart stabilization to the turn conformation. No regular repeating conformations were detected in the linker regions connecting each NPMeNA unit. Polyclonal antisera raised in rabbits against (NPMeNA)3 recognized intact P. falciparum sporozoites in an immunofluorescence assay as efficiently as antisera raised against (NPNA)3. This indicates that the type-I ,&turn detected in the PMecontaining peptide is closely related to the immunologically dominant portion of the folded CS protein. An improved knowledge of the three-dimensional structure of this protein may be of value for the design of second-generation synthetic malaria vaccines.