Successful vaccination against smallpox with conventional vaccinia virus is usually determined by the development of a vesicular skin lesion at the site of vaccinia inoculation, called a "take." Although previous vaccination is known to be associated with attenuation of the take, the immunology that underlies a no-take in vaccinia-naïve individuals is not well understood. We hypothesized that antibody profiling of individuals before and after receiving vaccinia virus would reveal differences between takes and no-takes that may help better explain the phenomenon. Using vaccinia virus proteome microarrays and recombinant protein enzyme-linked immunosorbent assays (ELISAs), we first examined the antibody response in vaccinia-naïve individuals that failed to take after receiving different doses of the replication-competent DryVax and Aventis Pasteur (APSV) smallpox vaccines. Most that received diluted vaccine failed to respond, although four no-takes receiving diluted vaccine and four receiving undiluted vaccine mounted an antibody response. Interestingly, their antibody profiles were not significantly different from those of controls that did show a take. However, we did find elevated antibody titers in no-takes prior to receiving DryVax that were significantly different from those of takes. Although the sample size studied was small, we conclude the failure to take in responders correlates with preexisting immunity of unknown etiology that may attenuate the skin reaction in a way similar to previous smallpox vaccination.