Insertional mutagenesis was performed with Tn1545 in the genetic background of an inIAB deletion mutant to identify new adhesion determinants in Listeria monocytogenes. Four insertion mutants defective in adhesion to eukaryotic cells were identified. Insertion sites were cloned by inverse-PCR and sequenced. The genetic organization of insertion regions was further analysed by screening and sequencing DNA fragments from a HindIII library and by searching databases. Three adhesion-defective mutants each had one copy of Tn1545 inserted into their chromosome. The insertion sites were different in the three mutants: (i) upstream from two ORFs in tandem, similar to dfp and priA of Bacillus subtilis, respectively; (ii) within an ORF encoding a putative 126 amino-acid-polypeptide with no significant similarity to any known protein; (iii) within an ORF similar to a B. subtilis ORF with no known function, just upstream from an operon similar to an ABC (ATP-binding cassette) transporter operon from B. subtilis. The excisants obtained from these mutants using the excision reporter plasmid pTCR9 recovered full adhesion capacity. A fourth mutant was the most severely defective in adhesion. It had five Tn1545 insertions, one of which was upstream from dfp and priA, and another of which was upstream from ami, a gene encoding a surface-exposed autolysin with a C terminus similar to that of InIB. Ami was clearly involved because an ami null mutant constructed in an EGDdeltainIA-F background was adhesion-defective. Thus new regions involved in the adhesion of L. monocytogenes to eukaryotic cells were identified. Further study is required to define more accurately the roles of these regions in the adhesion process itself.