We examined the lateral diffusion of I-A d on A20 cells following the binding of ovalbumin-derived peptides. The peptides were OVA 3 2 3 - 3 3 9 and OVA 3 2 5 - 3 3 5 and a related peptide OVA 3 2 5 - 3 3 5 S substituted H331Q. Only OVA 3 2 3 - 3 3 9 and OVA 3 2 5 - 3 3 5 were effectively presented by A20 cells to DO-11.10/S4.4 T cells as assessed by IL-2 production. Fluorescence photobleaching recovery (FPR) measurements showed anti-I-A d to have a lateral diffusion coefficient on untreated A20 cells of 1.8 ± 1.0 10 - 1 0 cm 2 s - 1 at 25°C with fluorescence recovery after photobleaching greater than 50%. After 24 h incubation of A20 cells with OVA 3 2 3 - 3 3 9 or OVA 3 2 5 - 3 3 5 , a subpopulation of A20 cells appeared that were approximately half the size of untreated A20 cells. Culture of A20 with OVA 3 2 5 - 3 5 5 S did not stimulate DO-11.10 cells or induce a size change in A20 cells. Class II molecules were laterally immobile on these small cells with fluorescence recoveries after photobleaching of less than 20%. The relative number of small cells in the A20 cell population was correlated with the immunogenicity of the peptides. These results suggest that immobilization of surface I-A d may be an important event in antigen presentation.