The aim of the present study was to evaluate the effect of different vitrification protocols on reactive oxygen species (ROS) and apoptosis in human ovarian tissue. Human ovarian tissue pieces were exposed to different vitrification solutions. The intracellular redox state level was measured using the fluorescent dye dichlorodihydrofluorescein diacetate. Imaging of apoptotic cells was monitored by anti-caspase-3 immunolabelling after vitrification and warming. Following equilibration in either 40% ethylene glycol (EG) (v/v), 0.35 M sucrose + 10% egg yolk extract (v/v) or 40% EG (v/v), 18% Ficoll-70 (w/v) + 0.35 M sucrose for 6 min, ovarian pieces were cooled to -196 degrees C using four different protocols. Tissue that was cooled very rapidly (plunged directly into liquid nitrogen in straws or on grids or plunged directly into metal filings precooled to -196 degrees C) showed no statistically significant increase in either tissue ROS levels or the number of apoptotic cells after warming. In contrast, cooling using a less rapid method (nitrogen vapour at -120 degrees C) resulted in significantly elevated ROS levels and apoptosis after warming. There were no significant differences between the two vitrification solutions. This indicates that human ovarian tissue pieces should be vitrified using very rapid cooling rates.