A monoclonal antibody based competitive inhibition assay was used to detect antibodies in horse sera to purified flagellar antigens from distinct Clostridium piliforme isolates. Sequential absorption of hyperimmune rat serum to C. piliforme isolate E (horse-origin isolate), a positive C. piliforme-immune horse serum, and other suspected immune horse sera with unrelated bacteria or C. piliforme isolates E or isolate R1 (rat-origin isolate) alone demonstrated the specificity of this assay for C. piliforme. This specificity was associated with the inhibition of monoclonal antibody binding to C. piliforme flagella, rather than to C. piliforme somatic antigens, by horse immunoglobulins partially purified from serum. Thirty seven of 162 horse sera possessed large amounts of antibody to the flagella of C. piliforme isolate E and 23 of the 162 had large amounts of antibody to the flagella of C. piliforme isolate R1; 9 of the sera possessed large amounts of antibody to both flagellar antigens. Absorption of these sera with isolate E or R1 demonstrated that antibody reactivity to the 2 C. piliforme isolates was isolate-specific and not due to antibody cross-reactive with both isolates. These results suggest that infection of horses with C. piliforme may be relatively common; and that they are susceptible to at least 2 distinct strains.