We have expressed in Escherichia coli the nonrepetitive repeat zone of the MSA-1 surface protein of the RO-33 isolate of Plasmodium falciparum. The recombinant protein was used to immunize mice and the resulting RO-33 monospecific serum was used to screen our P. falciparum strain collection in order to recover additional alleles lacking tripeptide repeats in block 2 of MSA-1. Only 1 (RO-71) out of 30 isolates tested reacted strongly with the serum by indirect immunofluorescence assay. Surprisingly, block 2 of the RO-71 MSA-1 allele contains tripeptide repeats resembling those of the K1 isolate of P. falciparum. Additional sequence analysis of the entire DNA coding for the 80-kDa MSA-1-derived surface component did not reveal any amino acid stretches similar to block 2 of RO-33 which could rationalize the immunological cross-reactivity. We eliminated the possibility that the RO-71 culture was contaminated with RO-33 type alleles of MSA-1 by Southern blotting and PCR analysis. The RO-33-specific mouse serum used for the initial selection of RO-71 did not react with the antigen in the denatured state (Western blot). This and the sequence analysis suggest that the cross-reactive epitope in the MSA-1 protein of RO-71 is conformational. The possibility that a truncated frame-shift protein encoded by mutated MSA-1 mRNA is recognized by the serum is discussed.