Background: Optimization of assisted conception outcomes involves the development of rapid, safe, effective techniques for the isolation of functional human spermatozoa free from significant DNA damage. In this study we describe a novel electrophoretic sperm isolation technique that achieves these objectives. Methods: The separation system consisted of a cassette comprising two 400 mu l chambers separated by a polycarbonate filter containing 5 mu mol/l pores and bounded by a 15 kDa polyacrylamide membrane to allow the free circulation of buffer. Semen was introduced into one chamber, current applied (75 mA at variable voltage) and within seconds a purified suspension of spermatozoa could be collected from the adjacent chamber. These cells were assessed for their count, viability, motility, morphology and DNA integrity. Results: The suspensions generated by the electrophoretic separation technique contained motile, viable, morphologically normal spermatozoa and exhibited low levels of DNA damage. Moreover, these cell suspensions were free from contaminating cells, including leukocytes. The technique was comparable to discontinuous gradient centrifugation except that it took a fraction of the time and generated cells with significantly less DNA damage. Conclusion: Electrophoretic separation represents a highly effective, novel approach for the isolation of spermatozoa for assisted conception purposes.