Hemagglutinating properties of Haemophilus paragallinarum serotype 2 and serotype C against freshly collected and glutaraldehyde (GA)- fixed chicken RBC were investigated. Different from serotype 1, the nontreated organisms of serotype 2 and serotype C lacked hemagglutinating activity. However, when the organisms were treated with potassium thiocyanate (KSCN) and/or sonication, activity occurred not only against GA-fixed chicken RBC, but also against GA-fixed RBC of various animal species. The maximum hemagglutination titer (1:64 to 1:256) was obtained against GA-fixed RBC with the KSCN-treated organisms that were also sonicated. The activity was inactivated by heating at 100 C or by treatments with formalin or trypsin, but not by treatments with hyaluronidase or neuraminidase. By using KSCN-treated and sonicated organisms and GA-fixed chicken RBC, a detection of hemagglutination-inhibition (HI) antibody was possible. The HI tests showed that serotype 2 hemagglutinin was immunologically distinct from serotype 1 and that the HI antibody correlated to protective activity against challenge exposure with serotype 2 and serotype C. Chicks having HI antibody greater than 1:5 were protected against challenge exposure with homologous type, but were not protected with serotype 1. Applicability of the HI test was also shown for evaluating protective potency in the serotype 2 vaccine, as well as in the serotype 1 vaccine.