The gene, flaA, encoding the flagellin protein of Listeria monocytogenes (strain 12067) has been isolated from an expression library in Escherichia coli using a flagellin‐specific monoclonal antibody. DNA sequence analysis of a positive clone revealed the presence of an open reading frame of 287 amino acid residues with a calculated molecular mass of 30.4 kDa. Comparison of this sequence with flagellins from other bacteria showed a significant degree of homology in both the N‐ and C‐terminal parts of the protein. The flagellin mRNA was determined to be 1 kb in size, which is the expected size for a monocistronic mRNA, and the temperature‐dependent expression of flagellin was found to be regulated at the transcriptional level. Southern blot analysis, using the flagellin gene as probe, indicated that L. monocytogenes can be divided into two groups. These groups correspond to the flagellar antigens AB and ABC, respectively, as well as to the two types of L. monocytogenes based on the DNA sequence of the listeriolysin gene.