Microspheres made from poly (lactic/glycolic acid) polymers have been considered as a new delivery system for single-dose tetanus toxoid (TT) vaccines. One of the most critical properties of the proposed vaccines is the loading and distribution of TT as this will have a profound effect on immunogenicity. As the concentration of TT in microspheres is very low sensitive assay methods are required. An assay incorporating monoclonal antibody (MAb) recognizing a neutralizing epitope and cross-reacting with TT was developed (MAb capture ELISA) which provided a sensitivity of 0.001 Lf/ml. An extraction procedure was devised which did not destroy the antigenicity and gave a recovery of 90.6 ±3.39% when applied to different preparations. The extracted TT was then quantified by MAb capture ELISA which was estimated to be 250-fold more sensitive than single-site ELISA for toxoid. The loading of 20 microspheres preparations (12 filled and 8 placebo) was determined by both protein micro-BCA assay and the developed assay for TT. The TT content obtained for the 12 FIlled microspheres preparations from different sources varied up to 400-fold (range 0.01–4.0 Lf/mg microspheres). The utility of the MAb capture ELISA for detection of total antigenic content in microspheres was conFIrmed by the observation that the determined TT loading correlated with the theoretical loading predicted from the protein content for the best preparations. Preparations with high loading gave the greatest peak response. There was no relationship between dose and the in vivoimmunogenic response, suggesting that encapsulated vaccines with differential loading, release properties and presence of excipients will have different response curves in vivo. Hence, the present assay, when combined with informatation on toxoid release rate and presence and effect of excipients may be of value in predicting in vivoresponse.