The progress toward the development of a single dose tetanus vaccine has been limited by the poor stability of the protein antigen, tetanus toxoid (TT), during its encapsulation in, and release from, biodegradable polymer microspheres. To investigate alternative microencapsulation approaches that may improve the stability of TT under these conditions, a two-step microencapsulation method has been devised to form microcapsules which consist of: (a) forming microcores of TT in a hydrophilic support matrix by spray-congealing, followed by (b) coating the microcores with poly(lactide-co-glycolide) (PLGA) by an oil-in-oil solvent extraction method. Several protein stabilizers including gelatin (with or without poloxamer 188), dextran, sodium glutamate, and polyethylene glycol were examined as potential core-materials. Among them, gelatin was superior in its ability to impart stability to TT against heat and moisture-induced inactivation. Microcores of this latter stabilizer and TT were encapsulated in PLGA using the foregoing technique, which exposed the dry antigen to minimal water in order to prevent its irreversible inactivation during exposure to the organic solvent. The microencapsulation method resulted in minimal loss of antigenically active TT (approximately 10-20%). Microscopic analysis of the microcapsules following preparation showed the microcores to be fully encapsulated. However, microcapsules containing TT and gelatin released the active antigen nearly completely within one day. Fluorescence confocal microscopy revealed that the swelling of the hydrophilic core-material was responsible for the burst-release behaviour. Manipulation of the polymer coating could not slow down this 'explosion' of the microcapsules. TT-containing PLGA microcapsules have been prepared using a novel microencapsulation method, which retains an extremely high fraction of antigenically active TT. Hence, these mechanistic approaches may be useful in the development of effective single-dose vaccines.