In order to know the effect of in vitro passages on the pathogenicity of the Salmonella gallinarum strain INTA 91, a lyophilized culture was compared with the same strain recently isolated from a sick bird. The mean lethal dose (LD₅₀) of the orally administered lyophilized culture was determined as 2.04 × 10⁸ colony-forming units (CFU)/chicken. There was no correlation between the LD₅₀ dose and the degree of disease produced; doses 10 or 100 times higher than the calculated LD₅₀ did not produce a more severe disease. In trial 1, chickens were challenged with 1.02 × 10⁹ CFU per chicken (5LD₅₀) of the lyophilized strain and reached 52.2% mortality at the end of the assay. In trial 2, three different groups of chickens were infected with a recent isolate of the same strain: 2.04 × 10⁸ CFU/chicken, 4.1 × 10⁸ CFU/chicken, and 2.1 × 10⁹ CFU/chicken (i.e., 1LD₅₀, 2LD₅₀, and 10LD₅₀ of the dose calculated for the lyophilized strain, respectively). These chicken groups presented higher mortality rates (90%, 100%, and 95%, respectively) than previous trials, showing that the S. gallinarum strain used here increased its virulence by in vivo infected chicken passage. In all assays, the disease started after an incubation period of around 5–6 days. To obtain reliable and reproducible results in future challenge experiments, a fixed limited number of in vitro passages of the S. gallinarum strain must be determined.