Sialic acid levels of human sperm were estimated using newly developed sperm purification procedure (inner column method). This was used to estimate net sialic acid content of forward motile sperm of normal morphology without any contamination of the seminal plasma, other cellular components, and immature or abnormal sperm. The sialic acid content in purified sperm was approximately 40% of that in the washed (dilution-washing method) sperm. It is suggested that biochemical properties of human sperm should be reevaluated using the inner column purification procedure.