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 (∼ 10 μm and ∼ 50 μm). Balb c mice were injected subcutaneously with 5 Lf (∼ 15 μg) 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, ∼ 95% and 90% radioactivity disappeared from site of injection for soluble TT or blank MS mixed TT and AlPO₄ adsorbed TT, respectively, whereas ∼ 55% and 70% radioactivity disappeared from site of injection for MS of average size ∼ 50 μm and ∼ 10 μm, 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, ∼20% radioactivity stayed at the site of injection for MS injected mice up to 4 weeks. At all time points, large MS (∼ 50 μm) showed more radioactivity at the site of injection than small MS (∼ 10 μm). 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.