Function-oriented immunoassays, such as complement fixation and neutralization, are not commonly used in the study of the Lyme disease agent, Borrelia burgdorferi. To determine whether such assays provide information additional to matrix-based methods, such as ELISA, polyclonal antisera and monoclonal antibodies were examined for their abilities to agglutinate viable borreliae and inhibit their in vitro growth in microtiter plates. Different strains of B. burgdorferi in both high and low passage were examined, and the related spirochete Borrelia hermsii and antibodies to it served as controls. Agglutination and complement-independent inhibition of growth with polyclonal sera from rats, mice, and humans and with monoclonal antibodies to outer membrane proteins OspA and OspB was demonstrated. Growth inhibition was obtained with Fab fragments as well as with whole IgG molecules. In comparison with an ELISA using whole cells, the growth inhibition and agglutination assays were generally more specific.