Neonatal ZU.ICR mice were infected with trophozoites of Giardia lamblia clone GS/M-83-H7 expressing the variant surface protein (VSP) H7 and were subsequently investigated for their serum antibody response directed against VSPH7. Recombinant polypeptides, representing overlapping segments of VSPH7, and native Giardia proteins were used as antigenic reagents to examine the antigenic substructure of VSPH7 and the extent of antigenic variation in vivo. VSPH7 proved to be the predominant antigen of the parasite with respect to serum antibody reactivity. The data indicated that VSPH7 basically consists of two antigenically distinct parts: (i) a unique, variant-specific 314-amino-acid N-terminal region which elicits a low antibody response preferentially detectable during the early phase of the infection and (ii) a 171-amino-acid C-terminal region which elicits a high antibody response during the later phase or after resolution of the infection. The epitopes of the C-terminal region appear to be shared by other, as yet uncharacterized, variant antigens. The highly conserved 34-amino-acid stretch at the extreme C terminus of VSPH7 exhibited no immunoreactivity to any of the sera tested. Further investigations indicated that antigenic variation of the intestinal parasite population was associated with a diversification into at least six to nine new antigen types. These variant antigens were extremely heterogeneous in size ranging from approximately 50 to 115 kDa. None of these proteins shared antigenic epitopes with the 314-amino-acid N-terminal portion, but several of them cross-reacted with antibodies specific to the 171-amino-acid C-terminal portion of the VSP from the original inoculum.