The growth of Pasteurella multocida strain X-73 (serotype 1) and P-1059 (serotype 3) in vivo instead of in vitro resulted in the expression of additional antigens, as revealed by SDS-PAGE profile and western blotting of the outer membrane detergent-insoluble fraction (DIF-OM). Minor protein bands ranging from 58-138 kDa were expressed by both strains; major bands ranging from 35-64 and 35-43 kDa were expressed by X-73 and P-1059, respectively. Antigens at 35-39 kDa were dense and dominant. Treatment of DIF-OM from both strains with sodium periodate and proteinase K, but not with trypsin, reduced ELISA reactivity; this suggested that DIF-OM was composed of glycoprotein. Western blotting with heterologous antisera demonstrated antigenic cross-reactivity between strains X-73 and P-1059 grown in vivo. Inactivated vaccine prepared from X-73 grown in vivo protected chickens against challenge with X-73, P-1059 and strain P-1662 (serotype 4); a similar vaccine prepared from in-vitro culture, however, protected only against the homologous serotype. Moreover, antiserum against X-73 grown in vivo but not against the same strain grown in vitro gave considerable passive protection against challenge with the heterologous strains P-1059 and P-1662.