Substantial evidence has implicated a role for CD8+ T cells in protective immunity against tuberculosis. Recombinant vaccinia (rVV) expressing Mycobacterium tuberculosis (MTB) proteins could be used, both as tools to dissect CD8+ T cell responses and, in attenuated form, as candidate vaccines. A panel of rVV expressing immunoprotective secreted proteins of MTB have been constructed. An initial rVV employed the vaccinia early/late p7.5 promoter and a gene encoding the 6 kDa early secretory antigenic target 6 protein (ESAT-6). Fidelity of cloning was verified by sequencing. Southern blotting and reverse-transcription polymerase chain reaction. However, attempts to identify protein expression using immunoblotting were unsuccessful. In order to overcome problems of low level expression, three further strategies were employed. These included: (i) expressing three different MTB genes encoding the Antigen 85 (Ag85) complex (30-32 kDa), as well as ESAT-6, (ii) comparing p7.5 with the more powerful T7 promoter, and (iii) use of a eukaryotic signal sequence belonging to human tissue plasminogen activator (tPA). tPA fusions were created by modifying the insertion plasmids, pi 108 (p7.5) and pWR510 (T7), prior to gene cloning. Nineteen rVV were constructed. Protein expression was identified for all rVV except Ag85C constructs. Surprisingly, the T7 hybrid system did not improve protein expression. However, addition of the tPA signal sequence produced a marked enhancement of expression. Three higher molecular weight 85B glycoforms were expressed by rVV-tPA-85B, but not rVV-85B Invitro coupled transcription-translation, in the presence of microsomes, gave further support for tPA-mediated membrane translocation and glycosylation. rVV-tPA-85A was used to infect human autologous macrophage as target cells in a 6 hour cytotoxic T cell (CTL) assay. Preliminary results suggested specific lysis of target cells using live BCG-stimulated T cells. Thus, these rVV represent powerful tools to investigate the role of CD8+ T cells in immunity to tuberculosis. In addition, they offer a potential strategy towards designing a vaccine against tuberculosis.