Monoclonal antibodies (mAbs) were raised in mice against the synthetic peptide (NANP)40, consisting of 40 (NANP) repeats of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and characterized. (i) Five of these mAbs recognized the P. falciparum CS protein in western blot experiments and in immunofluorescence assays using different preparations of sporozoites. The remaining two mAbs (CT3.2 and CT3.3, both IgG1) gave negative results by both techniques. (ii) When the anti-(NANP)40 peptide mAbs were functionally tested in vitro to assess their ability to inhibit the attachment and penetration of the parasites into cultured human liver cells, six of them exhibited inhibitory activities ranging between 66 and 90%. CT3.2 mAbs, also, inhibited sporozoite attachment and penetration, despite the negative results by immunofluorescence and western blot experiments. However, when immunofluorescence was repeated in the presence of calcium, CT3.2 did reveal a positive recognition of P. falciparum sporozoites, suggesting that this mAb could recognize the (NANP) sequence when calcium was bound to the repetitive peptide. (iii) Furthermore, the binding of an anti-(NANP)40 IgM mAb (CT1) to the solid-phase peptide was not inhibited by preincubation of the peptide with a mAb against the P. falciparum CS protein. (iv) Finally, one anti-(NANP)40 IgG1 mAb (CT3.1) was unable to bind to the shorter (NANP)3 peptide, although it recognized the (NANP)40 peptide and the P. falciparum CS protein. The results presented here suggest that heterogeneous antibody populations are produced upon immunization of mice with (NANP)40 synthetic peptide and that epitopes different from those simply related to the linear (NANP) amino acid sequence are likely to be present in long (NANP)n constructs as well as in the repetitive domain of the P. falciparum CS protein.