Centrifugal counter-current distribution (CCCD) in an aqueous two-phase system was used to detect changes associated with maturation of ejaculated ram spermatozoa. Spermatozoa obtained from three successive ejaculates of rams maintained in abstinence for one, two and three days were fractionated by CCCD. The results show that these ejaculates are relatively enriched in a cell population which presents a very high enhanced affinity to the lower dextran-rich phase. This cell population is not associated with loss of acrosomal integrity. In addition, it tends to disappear with longer abstinence periods, or after successive ejaculations at the same abstinence period, strongly suggesting that it is composed of immature cells. Therefore, phase partitioning can detect surface changes accompanying sperm maturation and offers a new possibility for sperm quality analysis.