A collection of 27 monoclonal antibodies (MAbs) against the S-layer protein (P100) of Thermus thermophilus HB8 has been obtained. They have been classified according to their ability to recognize S-layer regions expressed in E. coli from plasmids containing different fragments of its coding gene, slpA. The accessibility of the binding sites in hexagonal, trigonal, or tetragonal assemblies of P100 was analyzed by enzyme-linked immunosorbent assays with six of these MAbs and their respective Fab fragments. When packed hexagonally as the native S-layer (S1 assemblies), only a small region located near the amino terminus of the P1OO was accessible. However, when P1OO was assembled into trigonal (pS2 assemblies) or tetragonal (S2 assemblies) arrays, most of the protein domains analyzed were easily detected, thus suggesting that P1OO is assembled in S2 and pS2 in a similar way and that these two arrangements are quite different from the S1 assembly. Relationships between accessibility and sequence predictions are discussed.