Three SV40 escape mutants were identified by selection in the presence of monoclonal antibodies with neutralizing activity. The VP1 amino acid alterations in these mutants were: (1) K73-->E (in loop BC); (2) D77-->E (in loop BC); (3) K171-->R (in loop EF); and (4) Q175-->H (in loop EF). These residues are clustered in close proximity to each other on the surface of the native capsid protein, strongly suggesting that they form a conformational epitope directly recognized by the neutralizing antibody. To our knowledge, the present study represents the first experimental mapping of a neutralization epitope of a polyomavirus family member. Structural information regarding the neutralization epitope should be useful for clarifying the extent of cross-reactivity exhibited by the humoral immune response towards related primate polyomaviruses (e.g., SV40, BKV, and JCV).