The use of intracellular single chain antibodies has recently emerged as a highly efficient method of downregulating or ablating protein expression. In this regard, we have demonstrated that a single chain antibody directed against the extracellular domain of the erbB-2 molecule causes a specific toxicity in erbB-2 positive tumor types. To further investigate the mechanism of this effect, we developed a second anti-erbB-2 sFv predicted to recognize an alternate extracellular epitope of the erbB-2 molecule. When produced as a secreted protein from the erbB-2 negative COS-1 cell line, this sFv binds specifically to erbB-2 positive cells, indicating that cellular machinery is able to produce a properly folded and functional sFv protein. However, by several assays, this sFv was shown to be unable to retain the erbB-2 protein within the ER. These negative results have implications for the evaluation and utilization of sFv knockout strategies in experimental contexts.