The permeability properties and the exclusion limits of the crystalline surface layers (S layers) of two selected strains of Bacillus stearothermophilus were investigated. Measurements were performed of passive solute uptake into the intracellular space of native or glutaraldehyde-treated sacculi. Native sacculi were prepared from whole cells by extracting the cytoplasmic membrane with Triton X-100 under conditions which preserved the integrity of the S layer and the peptidoglycan-containing layer. The permeability barrier was found to consist of three adjacent layers, namely, the S layer, the peptidoglycan-containing layer, and an incomplete S layer attached to the inner face of the peptidoglycan-containing layer. In glutaraldehyde-treated sacculi the peptidoglycan was digested after stabilizing the S-layer lattice by chemical cross-linking. The solutes selected for the uptake measurements were mannose, proteins, and dextrans of increasing molecular weights. The S layers of both strains allowed free passage for molecules with a molecular weight of up to 30,000 and showed sharp exclusion limits between molecular weights of 30,000 and 45,000, suggesting a limiting pore diameter of about 4.5 nm.