The role of S-layer proteins (SLP) on the Pb²⁺ sequestrant capacity by <i>Lactobacillus kefir</i> CIDCA 8348 and JCM 5818 was investigated. Cultures in the stationary phase were treated with proteinase K. A dot blot assay was carried out to assess the removal of SLP. Strains with and without SLP were exposed to 0–0.5 mM Pb(NO₃)₂. The maximum binding capacity (q_max) and the affinity coefficient (b) were calculated using the Langmuir equation. The structural effect of Pb²⁺ on microorganisms with and without SLP was determined using Raman spectroscopy. The bacterial interaction with Pb²⁺ led to a broadening in the phosphate bands (1,300–1,200 cm⁻¹ region) and strong alterations on amide and carboxylate-related bands (νCOO⁻ as and νCOO⁻ s). Microorganisms without SLP removed higher percentages of Pb²⁺ and had higher q_max than those bearing SLP. Isolated SLP had much lower q_max and also removed lower percentages of Pb²⁺ than the corresponding whole microorganisms. The hydrofobicity of both strains dramatically dropped when removing SLP. When bearing SLP, strains do not expose a large amount of charged groups on their surfaces, thus making less efficient the Pb²⁺ removal. On the contrary, the extremely low hydrofobicity of microorganisms without SLP (and consequently, their higher capacity to remove Pb²⁺) can be explained on the basis of a greater exposure of charged chemical groups for the interaction with Pb²⁺. The viability of bacteria without SLP was not significantly lower than that of bacteria bearing SLP. However, microorganisms without SLP were more prone to the detrimental effect of Pb²⁺, thus suggesting that SLP acts as a protective rather than as a sequestrant layer.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Criotecnología de Alimentos