This study was conducted to evaluate the influence of a vancomycin pretreatment on the ability of marker (nalidixic-acid resistant) Salmonella Enteritidis (SE^M), field Salmonella Enteritidis (SE^F), and marker Salmonella Typhimurium (ST^M) strains to colonize within the intestinal and reproductive tracts and translocate to other organs of leghorn laying hens. In each of three trials, caged laying hens (76, 26, and 33 wk of age) were divided into six groups designated to receive SE^M, SE^F, or ST^M, and half were pretreated with vancomycin (n  =  11–12 hens). Vancomycin-treated hens received 10 mg vancomycin in saline/kilogram body weight orally for 5 days to inhibit Gram-positive bacteria within the intestines. On Day 6, all hens were concurrently challenged by oral, intravaginal, and intracolonal routes with Salmonella and placed into separate floor chambers by Salmonella strain. Two weeks postinoculation, all hens were euthanatized and the ceca, spleen, liver/gall bladder (LGB), upper (URT), and lower (LRT) reproductive tracts, and ovarian follicles were aseptically collected, and analyzed for Salmonella. Results did not differ for the three hen's ages and were therefore combined. The vancomycin pretreatment also had no significant effect on the colonization ability of SE^M, SE^F, or ST^M, and therefore results were combined within Salmonella strain. The marker strain of Salmonella Enteritidis was recovered from 21% of ceca, 4% of LGB, 9% of LRT, and 17% of the fecal samples. The field strain of Salmonella Enteritidis was recovered from 88% of ceca, 96% of spleen, 92% of LGB, 30% of LRT, 4% of URT, 13% of follicle, and 42% of the fecal samples. The marker strain of Salmonella Typhimurium was recovered from 100% of ceca, 74% of spleen, 91% of LGB, 30% of LRT, 9% of URT, 9% of follicle, and 100% of the fecal samples. Among ceca, spleen, LGB, and fecal samples, SE^F and ST^M colonization was significantly greater than SE^M colonization. Overall prevalence of Salmonella in the reproductive tracts of challenged hens was relatively low, ranging from 4% to 30%.