Twenty-eight isolates of Pasteurella haemolytica from domestic sheep (n = 14 isolates) and bighorn sheep (n = 14 isolates) were evaluated for leucotoxicity against peripheral blood neutrophils of bighorn sheep by adding bacterial culture supernatants to bighorn sheep neutrophils in vitro. Leukotoxic isolates of P. haemolytica, defined as causing >50% neutrophil death as measured by release of lactate dehydrogenase into culture supernatants, were identified from eight of 14 domestic sheep isolates and from 0 of 14 bighorn sheep isolates. The in vitro assay of isolates of P. haemolytica may provide a valid predictive measure of strain virulence of P. haemolytica, and of potential pneumonic episodes in bighorn sheep populations.