To determine the content of mitochondrial DNA (mtDNA) in oocytes from a range of patients with fertilization success and failure. Analysis of mtDNA content in fertilized and unfertilized oocytes and embryos by real-time polymerase chain reaction (PCR). University hospital infertility and research center. Fifty-four women seeking treatment for infertility. None. A total of 142 fertilized and unfertilized oocytes were classified into three main groups. Group I consisted of 35 fertilized oocytes from 21 patients; group II, 65 unfertilized oocytes from 36 patients; and group III, 42 degenerate oocytes from 23 patients. Mitochondrial DNA content was determined by SYBR Green real-time PCR-based assay. The mean mtDNA copy number for the fertilized oocytes was 250,454, whereas for the unfertilized group it was 163,698. There were significant differences for mtDNA copy number between the male factor and female factor infertility unfertilized oocytes and between the unexplained infertility and female factor infertility groups. The mean copy number for the degenerate oocyte group was 44,629, which was significantly different from the other subdivisions in this group. Mitochondrial DNA content is critical to fertilization outcome and serves as an important marker of oocyte quality, explaining some cases of fertilization failure.