The localization and activity of mitochondria in hamster oocytes during maturation and fertilization were monitored by using three different fluorescent probes: rhodamine 123 (Rh123), MitoTracker Red (MT-Red) and 3,3′-dihexyloxacarbocyanine iodide (DiOC₆(3)). Oocytes aged in vivo were also examined. The germinal vesicle (GV) oocytes exhibited a range of mitochondrial organizations, from a uniform distribution to a more cortical restriction. Metaphase I (MI) and MII oocytes included numerous mitochondria, among which the fluorescence intensity increased two-fold over that of GV oocytes. About half (54.5%) of the MII oocytes exhibited a mitochondria free zone at the cortex. An approximately 10 % decline in the density of mitochondria was observed in the oocytes aged 10 h post-ovulation, despite no significant changes. After fertilization, mitochondria became progressively aggregated and localized in the perinuclear region of all eggs examined. These comparative studies show that the signals for Rh123 and MT-Red were virtually coincident, but that for DiOC₆(3) displayed wider areas of bright organizations regardless of the very low concentration used in this study (5 ng/ml). In addition, transition from a relatively homogeneous distribution of active mitochondria to perinuclear clustering may reflect energy production and utilization during oocyte maturation and fertilization in hamsters, as reported in other species.