Pathogenicity island 1 (SPI-1) located at centisome 63 of the Salmonella chromosome encodes a type III protein secretion system that is essential for its pathogenicity. The translocation of effector proteins through this system results in the stimulation of signalling events, leading to actin cytoskeletal rearrangements and nuclear responses. These cellular responses ultimately lead to bacterial uptake, production of proinflammatory cytokines in non-phagocytic cells and the initiation of programmed cell death in macrophages. The regulation of expression of components and substrates of this type III secretion system is complex and involves the activity of several specific transcriptional regulatory proteins encoded within SPI-1. Here, we describe two additional regulatory proteins, SprA and SprB, which are encoded within SPI-1. SprA and SprB exhibit significant sequence similarity to the AraC/XylS and the LuxR/UhaP family of transcriptional regulatory proteins respectively. Insertion mutations in sprA and sprB did not significantly affect the transcription of invasion-associated genes and, consequently, did not affect the ability of Salmonella typhimurium to gain access into host cells. However, expression of sprA from an inducible heterologous promoter resulted in increased expression of genes associated with the centisome 63 type III secretion system and increased the ability of S. typhimurium to enter into host cells. Further analysis demonstrated that SprA acts either upstream or at the same level as HilA in the SPI-1 transcriptional regulatory cascade.