Contact with bone marrow stromal cells is crucial for the normal growth and development of B-cell precursors. We have previously shown that human bone marrow stromal cell tyrosine kinase activity can be activated by direct contact with B-lymphoid cells (J Immunol 155:2359, 1995). In the present study, we show that increased tyrosine phosphorylation of focal adhesion kinase, paxillin, and extracellular-related kinase 2 (or p42 MAP kinase) accounted for the major changes occurring in stromal cell tyrosine phosphorylation after 5 to 10 minutes of contact with the RAMOS B-lymphoma cell line. Although adhesion of B-cell precursors to stromal cells is primarily mediated by very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1), VLA-4-deficient and adhesion-deficient RAMOS cells were equally capable of stimulating stromal cell tyrosine phosphorylation. Similar changes in the tyrosine phosphorylation pattern of stromal cells were induced by contact with normal human B-cell precursors and several other B-lineage cell lines. After 5 to 30 minutes of contact with stromal cells, no change in protein tyrosine phosphorylation was detected in RAMOS or normal human B-cell precursors removed from stromal cells. Pretreatment of stromal cells with cytochalasin D abrogated contact-mediated enhancement of stromal cell tyrosine phosphorylation, suggesting that an intact cytoskeleton was essential. These results suggest that B-cell contact activates stromal cell signaling cascades that regulate cytoskeletal organization and transcription, independent of the interaction mediated by VLA-4 and VCAM-1.