Clone 3 is a mouse pre-B cell line that cannot grow in standard tissue culture media but is immortalized by coculture with a bone marrow-derived feeder layer. In addition, clone 3 cells can be passaged indefinitely in recombinant interleukin-7 (IL-7) in the absence of a feeder layer if maintained at high cell density. Using monoclonal antibody to IL-7 and Transwells ligated to dialysis membranes, we have examined the relative contributions of IL-7, both endogenous and exogenous, and of "non-IL-7" feeder layer factors to growth promotion of clone 3 cells. There is synergy between feeder layers and exogenous IL-7 that is most marked when the latter is present in suboptimal concentrations. The synergizing activity is not neutralized by antibody to IL-7 and appears to be freely dialyzable. This "non-IL-7" effect is common to two different feeder layers, the one derived from bone marrow (3E) being an IL-7 producer, and the other, 3T3 fibroblasts, making no detectable IL-7. These experiments reveal a substantial contribution of the dialyzable moiety to the total feeder layer effect, and are the first to demonstrate cytokine-dependent low-molecular-weight synergy in a coculture. This demonstration is possible because the synergizing cofactor(s) can cross a semipermeable membrane whereas the cytokine and its neutralizing antibody cannot.