BACKGROUND Given the importance of timing of denudation and oocyte injection during assisted reproductive technology cycles, a study was undertaken of the impact of timing on the outcome of intracytoplasmic sperm injection in a cohort of patients from a private in vitro fertilization center. METHODS The study included patients who underwent fresh intracytoplasmic sperm injection-embryo transfer by a single embryologist and gynecologist over a two-year period (January 2008 to March 2010). The primary outcome measure was the number of mature oocytes, and clinical pregnancy was the secondary outcome measure. RESULTS There was no significant difference between the three groups with respect to female age, but Group 3 showed a higher mean number of cumulus oopherus (14.46 versus 14.19), mature oocytes at time of denudation (12.72 versus 11.71), metaphase II injected (13.04 versus 11.85), and fertilized oocytes (9.75 versus 8.25) than Group 2. Group 2 showed a higher number of cumulus oopherus (14.19 versus 13.11), mature oocytes at time of denudation (11.71 versus 9.37), metaphase II injected (11.85 versus 9.7), and fertilized oocytes (8.25 versus 6.86) than Group 1. The maturation index, as well as positive clinical pregnancies, was significantly higher in Group 2 than Group 1 and in Group 3 compared with Group 1. Comparing Group 2 and Group 3, there was no significant difference regarding clinical pregnancy rates. CONCLUSIONS It is preferable to allow an interval between oocyte retrieval and sperm injection.