The guinea-pig skintest was used to test the pathogenic character of nine Bordetella bronchiseptica isolates derived from a number of different animals. One isolate was non-pathogenic, one showed a doubtful reaction whereas the others were pathogenic. Analysis of the cell envelope protein patterns by sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed only minor differences. Two major protein bands and at least thirty five minor bands were observed. The major protein with an apparent molecular weight of 37000 has properties similar to those of pore proteins of Enterobacteriaceae. Lipopolysaccharides (LPS), analysed by the same technique, could be separated into two fractions, LPS-I and LPS-II. The electrophoretic mobilities of the LPS of the strains were indistinguishable from each other with the exception of that of the non-pathogenic strain, which also differed serologically from that of the other strains. All sera of animals which had been successfully vaccinated with whole B. bronchiseptica cells showed a positive reaction with LPS-I whereas antibodies against the 37000 dalton protein, LPS-II as well as against several other proteins, were also detected in several sera. These results strongly suggest that LPS-I is a protective antigen. LPS-I and the 37000 dalton protein are accessible to antibodies in whole cells. This protein as well as a 28000 dalton protein were found to be located at the cell surface using iodination of whole cells with Iodo-Gen.(ABSTRACT TRUNCATED AT 250 WORDS)