Sperm motility assessments were conducted as part of a longitudinal study of human semen characteristics. This report evaluated the variation of sperm motility characteristics within an ejaculate, between ejaculates from the same man, and between healthy men without known toxicant exposure. Semen samples were collected monthly for 9 months from 45 men without fertility problems or exposure to known reproductive toxicants. For each specimen obtained for the 45 men participating in the study, measurements of curvilinear velocity, straight line velocity, linearity, amplitude of lateral head displacement, and beat cross frequency were collected. The overall ages for all samples ranged from 14 to 118 minutes. From all of the sperm motility measurements taken, at least 90% of the variation was noted between cells within a semen sample. For all variables, the component of variation between subjects was the smallest percentage. At least 75% of the variation was observed between samples from the same individual when sample means were used in the nested analysis of variation.