In this study, the use of Ca2+/Mg2+-free medium for biopsy of human embryos at the 4- to 10-cell stage on the third day of development was evaluated. When compared with control medium containing normal concentrations of Ca2+ and Mg2+ ions, the use of Ca2+/Mg2+-free medium allows an easier removal of blastomeres as illustrated by a lower rate of cell lysis as well as by a shorter time needed to perform the procedure. Subsequent embryo development to the blastocyst stage is not affected by the choice of biopsy medium, not even when embryos are exposed to the medium for 45 min. The use of Ca2+/Mg2+-free medium thus allows for an easier biopsy procedure during pre-implantation genetic diagnosis, while it does not result in a loss of developmental potential of the embryo to the blastocyst stage.