The wide occurrence of molecular rearrangements associated with expression of specific members within multigene families led us to investigate whether this also happens during antigenic variation of malaria parasites. We have investigated here the Pf60 multigene family restriction patterns of four distinct variants of the Plasmodium falciparum Palo Alto line propagated in Saimiri monkeys. The O and its cloned Oc variant both express the O serotype, while the R variant (derived from O parasites) and the Vc variant (derived from Oc parasites) express distinct serotypes. We show that a specific modification of the restriction pattern could be associated with antigenic switching in this line. The DNA of the variants which expressed the O serotype (O and Oc) had a specific 5.5 kb Hind III/Eco RI restriction fragment which was absent from the R or Vc parasite DNA, whereas both R and Vc DNA presented a 3.5 kb Hind III/Eco RI restriction fragment, which was absent from the O and Oc parasites. These results indicate that both expression and silencing of the O serotype were associated with specific restriction patterns, suggesting that some molecular rearrangement or some modification of the DNA might control expression of the variant surface antigen in malaria parasites.