We have developed an experimental system for testing mathematical model predictions concerning escape of autocrine ligands into the extracellular bulk medium. This system employs anti-receptor blocking antibodies against the epidermal growth factor receptor (EGFR)/transforming growth factor alpha (TGF alpha) receptor/ligand pair. TGF alpha was expressed under the control of a tetracycline-repressed promoter, together with a constitutively expressed human EGFR in B82 mouse fibroblast cells. This expression system allowed us to vary TGF alpha synthesis rates over a roughly 300-fold range by adjusting tetracycline concentration. TGF alpha accumulation in the extracellular bulk medium was then measured as a function of cell density, TGF alpha synthesis rate, and anti-EGFR blocking antibody concentration. Consistent with model predictions, amounts of ligand in the medium on a per cell basis were found to diminish as cell density was increased but with reduced dependence on cell density at higher ligand synthesis rates. Similarly consistent with model predictions, higher ligand synthesis rates also decreased the effect of anti-receptor blocking antibodies. Our investigation has established that we can successfully analyze and understand autocrine ligand secretion behavior from the basis of our theoretical model.