The in vitro effects of antibodies, complement, and (or) macrophages on Treponema pallidum have been previously characterized using relatively simple systems of organisms incubated with the immune components. In vivo, the more complex environment may alter immune reactivity. Experiments were performed to determine whether immobilizing and neutralizing antibodies retained their effectiveness in a more complex environment involving cultured mammalian cells. Two different protocols were used. In protocol A treponemes and normal or immune serum were mixed and added immediately to the cultured cells. In protocol B treponemes were preincubated for 18 h with cultured cells to maximize treponemal attachment; then normal or immune serum was added. With both protocols, attachment of organisms resulted in less efficient immobilization and neutralization. In further experiments, cultured cells were disrupted with Triton X, leaving cytoskeletal remnants on the vessel surface. Identical immobilization and neutralization experiments were performed in the presence of these remnants. In contrast to the findings with viable cultured cells, treponemal attachment to these nonviable remnants did not effect either antibody reaction. Attached organisms were immobilized or neutralized just as efficiently as unattached organisms. Results are discussed in terms of the altered immune reactivity in more complex in vitro environments.