In most attenuated Salmonella enterica vaccines, heterologous antigens are expressed under the control of strong inducible promoters to ensure a high level of synthesis. Although high expression levels of the antigen can improve the immunogenicity of the vaccine, they might be toxic to the Salmonella carrier. Expression problems could be avoided by the use of promoters with specific characteristics with respect to strength and timing of expression. To study the expression of ten selected promoters, translational promoter-green fluorescent protein (GFP) fusions were analyzed in three attenuated Salmonella strains, Ty21a, SL3261 and PhoP ^C . Promoter expression was evaluated both in vitro and in intracellular conditions using flow cytometry and confocal microscopy, with specific focus on the levels and timing of expression. We identified one major candidate promoter (Pasr) that could be used to express antigens specifically during in vivo conditions, without impairing bacterial growth during in vitro vaccine production.