Regulated antigen expression can influence the immunogenicity of live recombinant Salmonella vaccines, but a rational optimization has remained difficult since important aspects of this effect are incompletely understood. Here, attenuated Salmonella enterica serovar Typhimurium SL3261 strains expressing the model antigen GFP_OVA were used to quantify in vivo antigen levels by flow cytometry and to simultaneously follow the crucial early steps of antigen-specific T-cell responses in mice that are transgenic for a T-cell receptor recognizing ovalbumin. Among seven tested promoters, P(pagC) has the highest activity in murine tissues combined with low in vitro expression, whereas P(tac) has a comparable in vivo and a very high in vitro activity. Both SL3261 (pP(pagC)GFP_OVA) and SL3261 (pP(tac)GFP_OVA) cells can induce potent ovalbumin-specific cellular immune responses following oral administration, but doses almost 1,000-fold lower are sufficient for the in vivo-inducible construct SL3261 (pP(pagC)GFP_OVA) compared to SL3261 (pP(tac)GFP_OVA). This efficacy difference is largely explained by impaired early colonization capabilities of SL3261 (pP(tac)GFP_OVA) cells. Based on the findings of this study, appropriate in vivo expression levels for any given antigen can be rationally selected from the increasing set of promoters with defined properties. This will allow the improvement of recombinant Salmonella vaccines against a wide range of pathogens.