Aims: To investigate the functional role of surface layer proteins (S-layer) in probiotic strain Lactobacillus acidophilus M92, especially its influence on adhesiveness to mouse ileal epithelial cells. Methods and Results: Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, ca at 45 kDa in L. acidophilus M92. Southern blot with pBK1 plasmid, containing slpA gene, gave a positive signal, suggesting that L. acidophilus M92 has a slpA gene coding for the S-layer proteins. S-layer proteins of this strain are present during all phases of growth. The S-layer proteins appeared when cells treated with 5 mol l(-1) LiCl were allowed to grow again. Removal of the S-layer proteins reduced adhesion of L. acidophilus M92 to mouse ileal epithelial cells. Furthermore, the viability of cells without S-layer were reduced in simulated gastric juice at low pH range (2, 2.5, 3) and simulated pancreatic juice with bile salts (1.5 and 3 g l(-1)). S-layer proteins of L. acidophilus M92 were resistant to pepsin and pancreatin, in contrast, the treatment with proteinase K led to a significant proteolysis of the S-layer proteins. Conclusions: These results demonstrated functional role of S-layer; it is responsible for adhesiveness of Lactobacillus acidophilus M92 to mouse ileal epithelial cells and has a protective role for this strain. Significance and Impact of the Study: S-layer proteins have an important role in the establishment of probiotic strain Lactobacillus acidophilus M92 in the gastrointestinal tract.