Many clinical programs in in vitro fertilization (IVF) use the classic Percoll sperm preparation technique to obtain a subpopulation of highly mobile spermatozoa. This Percoll discontinuous gradient centrifugation technique (densities ranging from 1.042 to 1.116 g/mL) was used at room temperature to separate human spermatozoa fractions in order to determine differences in chromatin stainability. The stainability was measured by analysis of DNA fluorochrome uptake by flow cytometry using human peripheral blood lymphocytes as an internal standard. A significant difference in chromatin stainability was noted between the residual fraction (immobile spermatozoa remaining at the top of the Percoll gradient) (44.2%) and the selected one (highly mobile spermatozoa harvested in the 90% and 100% steps) (36.3%). The sperm DNA heterogeneity, as defined by the coefficient of variation (CV), was significantly lower for the selected fraction as compared to the residual one (CV = 12.8% versus 15.4%). A marginal population of spermatozoa adjacent to the germinal peak was also identified. The percentage of this marginal population was significantly lower for the selected fraction as compared to the residual one (7.4% versus 22%). Using a biochemical in vitro decondensation method, the chromatin stainability of the selected and residual fractions (56.9% versus 62.9%) was also determined, demonstrating that the chromatin of the selected fraction was less stainable. This study confirmed that Percoll centrifugation selects a germinal population that is denser and less stainable than with other techniques and analyzed the relation between density, high mobility and probable capacitation.