Abstract: Capacitation and acrosome reaction (AR) of human spermatozoa are prerequisites for fertilization. Annexin-V-MACS is able to separate apoptotic from nonapoptotic sperm on the basis of their externalization of phosphatidylserine (EPS). The nonapoptotic (EPS−) fraction is characterized by the lowest amounts of membrane alterations, caspase activation, disrupted mitochondrial potential, and DNA fragmentation. The aim of our study was to investigate the separation effect of Annexin-V-MACS on capacitation and AR in nonapoptotic sperm. Semen specimens from 10 healthy donors were separated into 2 samples each, one was left untreated (control) and the second was subjected to Annexin-V-MACS. Two aliquots of both, the control as well as the EPS− fraction after Annexin-V-MACS, were incubated in HTF at 37°C, 5% CO2 for 3 h either with 3% BSA (capacitation) or without additives. Capacitation was monitored by tyrosine phosphorylation (TyrP) using Western blot technique. AR was determined by labeling with CD46-FITC before and after stimulation with calcium-ionophore A23187, followed by flow cytometric evaluation of the percentage of CD46+ sperm. Densitometric analyses of the 105-kDa and 80-kDa bands of the TyrP Western blots demonstrated highest TyrP in the capacitated EPS− aliquots. There was no difference in spontaneous AR in all groups. AR was best inducible in EPS-negative sperm after capacitation. Nonapoptotic human spermatozoa with intact plasma membranes are characterized by superior ability to capacitate and consequently by maximum potential to perform AR after stimulation. Selection of EPS-negative sperm may be of advantage for assisted reproduction in order to prepare the sperm subpopulation with the highest fertilizing potential.