The use of biodegradable polymer microspheres as a single dose vaccine delivery system was investigated by using tetanus toxoid (TT). In order to compare the immunogenicity of TT-microspheres (TT-MS) with aluminum hydroxide (alum)-based TT, BALB/c mice were immunized with TT in different formulations including individual or mixtures of MS and TT-alum. All TT-MS formulations elicited high proliferative and antibody responses comparable to those obtained with TT-alum formulation. Antibody levels remained elevated over a long period of time. Certain individual MS preparations elicited lower antibody titers than the MS mixtures. More importantly, the proliferative and antibody responses induced by a single injection of three TT-MS mixtures with different particle sizes and degradation rates were similar to those obtained with three injections of TT-alum. In addition, TT-MS induce similar isotypesubclass antibodies to those TT-alum induced. These results raise the possibility to obtain optimal and long-lasting immune responses by single administration of the three TT-MS mixture formulations alone.