Salmonella enterica serovar Typhimurium infection of chickens is a major public and animal health problem. In young chicks, S. Typhimurium infection results in severe systemic infection; in older chicks, infection results in prolonged gastrointestinal tract colonization. Here we determined the role of the Salmonella pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2) type III secretion systems in systemic infection and gastrointestinal tract colonization of the chicken though experimental infection of chicks with a S. Typhimurium strain with mutations in the genes encoding the secretion system machinery of SPI-1 (spaS) and SPI-2 (ssaU) that prevent secretion of effector proteins. In 1-day-old chicks, mutation of SPI-2 lead to a decrease in both systemic bacterial numbers and pathology, although no difference in gastrointestinal numbers was observed. Mutation of SPI-1 had little effect in 1-day old chicks. In 1-week-old animals the SPI-2 mutants could not be detected systemically and colonized the gastrointestinal tract only in low numbers in comparison with the parent strain, and was cleared in 1 week. The SPI-1 mutant showed greatly reduced levels of systemic infection, and colonized the gastrointestinal tract at a lower level than the parent strain. The findings show that the SPI-2 type III secretion system is required for systemic S. Typhimurium infection in both infection models, and that it plays a significant role in gastrointestinal colonization. The SPI-1 system is involved in both systemic infection and gastrointestinal colonization, but does not appear absolutely essential for either infection process.