We have identified a novel mAb, SG31, which recognizes the mouse integrin α4 subunit. Unlike the epitopes recognized by other anti-α4 antibodies, the SG31 epitope is expressed on subpopulations of thymocytes and peripheral T cells. After manganese ion, but not phorbol myristic acetate activation, the epitope is induced and expressed on the majority of peripheral T cells. These data suggest that the SG31 epitope is an activation epitope and that manganese ions activate α4 integrins by inducing a conformational change. Comparative flow cytometric analyses showed that the SG31 epitope as well as the epitope detected by other anti-α4 antibodies is expressed on all B lineage cells. In the T lineage, expression of the α4 integrins is down-regulated during thymocyte development. Although mature thymocytes still express the α4 integrins, they lose almost entirely the activation epitope recognized by SG31. In contrast, the most immature thymocytes express high levels of this epitope. In the periphery, SG31 epitope is expressed mostly by activated T cells, in contrast to the overall population of T cells that express the α4 integrins at homogenous levels. These results suggest that the activation of the α4 integrins is parallel to that of T cells.