The interaction between pathogenic mycobacteria and epithelial cells seems to play an important role during early stages of tuberculosis and leprosy, two important mycobacterial diseases. Previous studies on the molecular mechanisms of this interaction have identified the protein Hlp/LBP as a major bacterial adhesin, mediating bacterial binding to glycosaminoglycans (GAGs) present on the host cell surface. The binding site in Hlp/LBP to heparin was mapped at the N - terminal region (residues 31-50) of protein by Aoki et al. (J Biol. Chem. 279(38):39798-806, 2004). However, in contrast to these results, using truncated versions of the protein, we  have recently mapped  the alanine/lysine/proline rich repeats present in the C - terminal region of Hlp/LBP as a major  binding site of the protein for Heparin/Heparan Sulfate (Soares de Lima  et al., Microbes Infec. 7(9-10):1097-109, 2005).  In order to clarify these conflicting results, in the present study we used a panel of 30 mer synthetic peptides covering the full length of the protein to define the binding site(s) of Hlp/LBP to GAGs. The capacity of these peptides to bind to Heparin/Heparan Sulfate was analyzed employing three distinct assays: i) solid phase assays in microplates; ii) Heparin-Sepharose affinity chromatography; and iii) Nuclear Magnetic Resonance. The results here obtained indicate that Hlp/LBP interacts with GAGs through multiple sites. Besides the 31-50 binding site previously described, a second region including the Heparin-binding consensus sequence (residues 52 to 59)  and the DNA-binding consensus sequence (residues 46 to 65) at the N-terminal region of Hlp/LBP was shown to interact with Heparin and Heparan sulfate. Additionally, the repetitive alanine/lysine/proline sequences were confirmed as a third Heparin-binding site localized at the C-terminal half of Hlp/LBP. Moreover, flow cytometry assays were performed to show that the pre-incubation of mycobacterial cells not expressing Hlp/LBP with a pool of synthetic peptides corresponding to the Heparin-binding sites of the protein was capable of restoring their capability of adhesion to host cells. Our data indicate that, similarly to the prerequisites previously defined for the binding of Heparin to other proteins, the presence of positively charged residues in conjunction with neutral/hydrophobic amino acids, and with a defined spatial conformation constitute important features for the interaction of Hlp/LBP with GAGs.