Cell surface hydrophobicity is one of the most important factors controlling adhesion of microorganisms to surfaces. In this paper, cell surface properties of lactobacilli and recombinant lactobacilli with and without a surface layer protein (SLP) associated with cell surface hydrophobicity were determined, including water contact angles, zeta potentials as a function of pH, the nitrogen contents of the cell surface and adhesion to hexadecane. Two strains possessing an S-layer (Lactobacillus acidophilus ATCC4356 and L. crispatus JCM5810) showed the highest water contact angles (76 and 55°, respectively) and the highest N/C surface concentration ratios by X-ray photoelectron spectroscopy (0.172 and 0.160, respectively), indicative of the presence of S-layer proteins. L. casei 393*/CA5′A, with the SLP of L. crispatus JCM5810 anchored to its surface had higher water contact angles (62°) than its parent strain (32°), but no higher amount of cell surface nitrogen. However, anchoring of the SLP did stimulate its adhesion to hexadecane. LiCl treatment, removing S-layer and other non-covalently linked surface proteins, increased water contact angles and N/C ratios for L. crispatus JCM5810 and L. casei 393*/CA5′A, while L. acidophilus ATCC4356 showed a decrease in the N/C ratio like for L. gasseri LMG9203, that lacks an SLP. The isoelectric point of all but one Lactobacillus strain varied between 3.2 and 4.6, whereas strain L. crispatus JCM5810 was positively charged over the entire pH range. A hierarchical cluster analysis, using all cell surface hydrophobicity associated properties as input, yielded one cluster for strains possessing SLP, well separated from the other strains, including strains secreting SLP. It is concluded that SLP conveys hydrophobicity to the Lactobacillus cell surface and enhances its adhesion to hexadecane through hydrophobic interactions.