Lymphoid follicles of the ileal Peyer's patch (PP) of young sheep function as the major source of B cells and a site of immunoglobulin (Ig) receptor diversification. However, extensive cell death in culture has restricted investigations of ileal PP follicular (iPf)B cell biology. We investigated the possibility that sustained iPfB cell proliferation may require an interaction with mesenchymal stromal cells (SC). Four SC lines, cloned from lymphoid follicles of the ileal PP, and various sheep and xenogeneic mesenchymal cells were used to characterize the nature of iPfB cell-SC interactions. A sustained proliferative response was unique to iPfB cells, required iPfB cell-SC contact, and SC membranes functioned as intact SC to either enhance or inhibit iPfB cell proliferative responses. The iPfB cell proliferation in SC co-cultures was accompanied by extensive cell death and a slow decline in viable cell number. Flow cytometric analysis confirmed that viable lymphocytes, present in SC co-cultures, were immature B cells that expressed surface IgM, with either lambda or kappa. Ig light chain, and that SC co-culture inhibited iPfB cell differentiation. Finally, addition of soluble anti-sheep Ig to iPfB cell-SC co-cultures did not inhibit SC-dependent iPfB cell proliferation or iPfB cell binding to SC. These data indicate that an interaction between specific SC membrane molecules and non-Ig molecules of iPfB cells either supported or inhibited a self-renewing proliferative response by immature (sIgMLo, BAQ44A-) iPfB cells. Finally, SC-dependent iPfB cell proliferation was independent of T cells and extrinsic antigen which further suggests that a functionally distinct B cell population resides in lymphoid follicles of the ileal PP.