Radiolabeled tetanus toxoid (TT) was prepared by detoxifying chromatographically purified tetanus toxin with 14C-labeled formaldehyde. 14C-TT was encapsulated inside poly (D,L-lactide-co-glycolide, 50/50) microspheres (MS) of varying average size (approximately 10 microns and approximately 50 microns). Balb/c mice were injected subcutaneously with 5 Lf (approximately 15 micrograms) of 14C-TT, encapsulated in MS, mixed with blank MS without encapsulated antigen, as soluble antigen or adsorbed onto aluminum phosphate (AlPO4) and radioactivity was monitored at the site of injection, draining lymph nodes, blood, liver, spleen, and kidneys at various intervals. At one day, approximately 95% and 90% radioactivity disappeared from site of injection for soluble TT or blank MS mixed TT and AlPO4 adsorbed TT, respectively, whereas approximately 55% and 70% radioactivity disappeared from site of injection for MS of average size approximately 50 microns and approximately 10 microns, respectively. By 7 days, 99% of radioactivity disappeared from site of injection for soluble TT or blank MS mixed TT, whereas 2-3% radioactivity persisted at the site of injection for AlPO4 adsorbed TT for 4 weeks. In contrast, approximately 20% radioactivity stayed at the site of injection for MS injected mice up to 4 weeks. At all time points, large MS (approximately 50 microns) showed more radioactivity at the site of injection than small MS (approximately 10 microns). Other organs showing radioactivity were draining lymph nodes and kidneys. Small MS with encapsulated TT showed highest level of radioactivity in lymph nodes at 4 h. In kidneys, soluble and AlPO4 adsorbed TT showed a peak of radioactivity at 4 h whereas TT encapsulated in MS showed a peak of radioactivity at 7 days. These results indicate that AlPO4 did not act as a depot for TT at the site of injection, but TT encapsulated in MS did form a depot for approximately 1 month.