We examined the effects of simulated stress on susceptibility of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) neutrophils to Pasteurella haemolytica leukotoxin in a blocked, crossover experiment. Ten captive-raised bighorn sheep were sampled 10 hr after separate administrations of long-acting adrenocorticotrophic hormone (ACTH) gel and normal saline (control). We then compared in vitro leukotoxin-dependent neutrophil death rates after exposure to culture supernatants from four unique P. haemolytica isolates (one from domestic and three from bighorn sheep). Simulated stress effects were evidenced by elevated (P = 0.002) mean plasma Cortisol concentrations, more neutrophils (P = 0.037), and fewer lymphocytes and eosinophils (P ≤ 0.043) in ACTH-treated bighorn sheep. Maximum leukotoxin-dependent neutrophil death rates were ≥61% for three of four P. haemolytica isolates tested. For all three cytotoxic isolates, neutrophil death rates at 150 μg/50 μl supernatant were about 1.13 times higher (P = 0.0001) after bighorns received ACTH; for two of these, overall neutrophil death rates were higher (P ≤ 0.001) in ACTH-treated bighorn sheep. Although variable leukotoxin production among P. haemolytica strains appeared principally responsible for differences in leukotoxin-dependent neutrophil death rates, susceptibility of bighorn sheep neutrophils to leukotoxin was increased by prior exposure to elevated plasma Cortisol concentrations. It follows that if similar processes occur in neutrophils and alveolar macrophages in vivo, they could contribute to greater susceptibility of stressed bighorn sheep to pneumonic pasteurellosis.