A pilot study was performed to test the diagnostic value of in vitro DNA fluorescence in oocytes that failed to fertilize after IVF. Ten patients with a cleavage rate less than 20% after IVF were included.Uncleaved oocytes were observed by fluorescence microscopy after incubation with the DNA fluorescent dye Hoeschst 33342. Four main causes which may have contributed to the low cleavage rate were found: (1) sperm incapacity to penetrate the oocyte despite the absence of the usual criteria for male infertility, (2) oocyte immaturity, (3) delayed fertilization, and (4) oocyte abnormalities revealed by aberrations in the morphology of the female chromatin.The possibility of a rapid and detailed analysis of the maturational status of unfertilized oocytes, the morphology of the female chromatin, the presence and quantity of spermatozoa tightly bound to the zona pellucida, and sperm penetration into the oocyte without subsequent pronucleus formation, using DNA fluorescence, allows us to clarify further the cause of fertilization failure and to orient infertility treatment toward the male, the female, or both partners.