Alum adjuvants have been in continuous clinical use for more than 80 yr. While the prevailing theory has been that depot formation and the associated slow release of antigen and/or inflammation are responsible for alum enhancement of antigen presentation and subsequent T-and B-cell responses, this has never been formally proven. To examine antigen persistence, we used the chimeric fluorescent protein EαGFP, which allows assessment of antigen presentation in situ, using the Y-Ae antibody. We demonstrate that alum and/or CpG adjuvants induced similar uptake of antigen, and in all cases, GFP signal did not persist beyond 24 h in draining lymph node antigen-presenting cells. Antigen presentation was first detectable on B cells within 6–12 h of antigen administration, followed by conventional dendritic cells (DCs) at 12–24 h, then finally plasmacytoid DCs at 48 h or later. Again, alum and/or CpG adjuvants did not have an effect on the magnitude or sequence of this response; furthermore, they induced similar antigen-specific T-cell activation in vivo. Notably, removal of the injection site and associated alum depot, as early as 2 h after administration, had no appreciable effect on antigen-specific T-and B-cell responses. This study clearly rules out a role for depot formation in alum adjuvant activity.—Hutchison, S., Benson, RA, Gibson, VB, Pollock, AH, Garside, P., Brewer, JM Antigen depot is not required for alum adjuvanticity.