The colonization of intestinal and systemic tissues by Salmonella enterica serovars with different host specificities was determined 7 days after inoculation of 1 to 2-month-old lambs. Following oral inoculation, S. enterica serovars Abortusovis, Dublin, and Gallinarum were recovered in comparable numbers from the intestinal mucosa, but serovar Gallinarum was recovered in lower numbers than the other serovars from systemic sites. The pattern of bacterial recovery from systemic sites following intravenous inoculation was similar. The magnitude of intestinal invasion was evaluated in ovine ligated ileal loops in vivo. Serovars Dublin and Gallinarum and the broad-host-range Salmonella serovar Typhimurium were recovered in comparable numbers from ileal mucosa 3 h after loop inoculation, whereas the recovery of serovar Abortusovis was approximately 10-fold lower. Microscopic analysis of intestinal mucosae infected with serovars Typhimurium and Dublin showed dramatic morphological changes and infiltration of inflammatory cells, whereas mucosae infected with serovars Abortusovis and Gallinarum were indistinguishable from uninfected mucosae. Together these data suggest that Salmonella serovar specificity in sheep correlates with bacterial persistence at systemic sites. Intestinal invasion and avoidance of the host's intestinal inflammatory response may contribute to but do not determine the specificity of serovar Abortosovis for sheep. Intestinal invasion by serovar Abortusovis was significantly reduced after mutation of invH but was not reduced following curing of the virulence plasmid, suggesting that the Salmonella pathogenicity island 1 influences but the virulence plasmid genes do not influence the ability of serovar Abortusovis to invade the intestinal mucosa in sheep.