Objective: Evaluate sperm chromatin stability and its relationship with the rate of fertilization after procedures of intracytoplasmic sperm injection (ICSI) in a program of assisted reproduction. Design: Prospective study. Setting: Institute of Gynecology and Reproduction. Patients: Thirty-three women with their respective partners (12 couples in the study group and 21 couples in the control group) participating in a program of assisted reproduction. The study group was defined as men with >30% of non-decondensed spermatozoa (high sperm chromatin stability). Interventions: A part of each seminal sample was used to evaluate sperm chromatin stability under SDS and EDTA treatment and the second aliquot was used for the ICSI procedure. Fertilization was evaluated 16-18 h post sperm injection at a pronuclear stage. The fertilized oocytes were further cultured for 24-48 h before transfer to the patient. Main outcome measures: Fertilization rate. Results: Thirty-five oocytes (35.7%) in the group of study and 109 oocytes (78.9%) in the control group showed two pronuclei (P<0.001). The coefficient of determination between the SDS+EDTA (Grade 2) and rate of fertilization was r 2=0.85 (P<0.001) and the coefficient of regression was 1.72±0.19 (β±ES) (P<0.001). Conclusions: High sperm chromatin stability is a factor which reduces the rate of fertilization after ICSI procedure. © 2007 Springer.