The production of the K99 antigen by enterotoxigenic Escherichia coli strains with various O antigens was investigated by means of slide agglutination tests, enzyme-linked immunosorbent assays, and hydrophobic interaction chromatography. The extent of K99 production appeared to be dependent on the nutrient medium, as well as on the incubation temperature. Minimal salt medium with glucose and semisynthetic Minca medium were the most suitable for K99 production. In complex media the production of K99 antigen was strongly reduced. Optimal amounts of K99 antigen were produced at 37 degrees C. At 30 degrees C, weak production of K99 antigen was detected by hydrophobic interaction chromatography of enzyme-linked immunosorbent assay. Slide agglutination tests were negative with cultures grown below 32 degrees C. The production of K99 antigen appeared to be related to the O antigen carried by the host strain, but it seemed to be independent of the absence or the presence of various K polysaccharide antigens. Under all conditions used, strains with antigen O101 produced about 10 times more K99 antigen than did strains with antigen O8, O9, or O20. Transfer of the K99 plasmid from wild-type strains of different O antigens to E. coliK-12 C600 confirmed that phenotypic expression of the K99 antigen is most probably related to the cell wall composition of the host.