Magnetic-activated cell sorting (MACS) using paramagnetic annexin V-conjugated microbeads eliminates spermatozoa with externalized phosphatidylserine, which is considered one of the features of apoptosis. The objective of this study was to evaluate sperm recovery following the use of MACS as a sperm preparation technique. Mature spermatozoa were separated and divided into two fractions: the first was prepared by density gradient centrifugation (DGC) and MACS, while the second was prepared by DGC only. Following MACS, the percentage of cells collected in the annexin-negative fraction was significantly higher than the annexin-positive fraction and the sperm recovery rate was 73.8 +/- 12.1%. In conclusion, the integration of MACS with DGC can be considered as an effective sperm preparation technique that does not lead to significant cell loss. Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize the outcome of assisted reproduction.