The eradication of smallpox by vaccination with vaccinia virus was probably one of the greatest achievements of vaccinology. However, the immunological basis of this protection is not fully understood. To this end, we have used protein microarrays of the vaccinia (Western Reserve, WR) proteome to profile antibody reactivities after primary infection or boosting with the licensed smallpox vaccine, Dryvax, and with archival convalescent smallpox sera. Some 25 antigens were consistently recognized by Dryvax sera, of which half were envelope proteins (notably, H3, A13, B5, and D8). The remainder consisted mainly of core proteins (e.g. A10, L4, and I1), proteins involved in intracellular morphogenesis (A11, D13), and the A-type inclusion protein, WR148. Convalescent smallpox sera also detected vaccinia antigens on the array, consistent with the notion that there is serological cross-reactivity between these two orthopox species that underlies protection. Moreover, the profiles of immunodominant antigens recognized by variola-infected individuals and Dryvax vaccinees were indistinguishable. This is the first description of antibody-specificity profiles induced after smallpox infection. The array data indicate that a significant component of the antibody response is not involved in virus neutralization, although these antigens should be considered alongside the envelope proteins as potential candidates for diagnostic and vaccine applications.