The molecules effecting adhesion of acute lymphoblastic leukemia (ALL) cells are not well defined. We investigated the expression of very late activation (VLA) integrins in five human leukemic cell lines of pre-B cell phenotype. VLA-4 was found to be the dominant integrin in all five, three possessed VLA-5, and one VLA-6. None had VLA-2, or VLA-3. Since certain anti-VLA-4 monoclonal antibodies (mAb) have been reported to induce homotypic aggregation of T and B lymphocytes we investigated the possibility that VLA-4 might be involved in aggregation of pre-B cells. mAb 44H6 (anti-VLA-alpha 4), and 4B4 (anti-VLA-beta 1) induced strong aggregation which was not blocked by the anti-FC gamma IIR mAb IV.3. However, aggregation was effected in only three of the five lines suggesting the involvement of molecules other than VLA-4. The level of expression of CD9, but not that of CD11a, CD18, CD19, CD44, or CD54, was found to correlate with the level of aggregation. Of mAb directed to CD9, CD19, CD44, endoglin, and HLA-DR only mAb to CD9 induced aggregation. Admixture of mAb ALB6 (anti-CD9) and mAb 44H6 neither potentiated nor inhibited the response indicating a common effector mechanism. We suggest that the level of CD9 may determine the level of VLA-regulated adhesion, and therefore the adhesive phenotype of leukemic pre-B cells.