BACKGROUND: Failed fertilization occurs in 2–3% of ICSI cycles and is mainly due to lack of oocyte activation. Heterologous ICSI of patient's sperm in mouse oocytes allows discrimination between sperm- and oocyte-related aetiologies of activation failure. Assisted oocyte activation (AOA) by Ca-ionophore treatment can initiate fertilization in subsequent therapeutic ICSI. We report on diagnosis and clinical treatment in 17 patients with previously failed fertilization. METHODS: Sperm from patients were injected into mature mouse oocytes. Activation capacity was assessed by 2-cell formation (mouse oocyte activation test, MOAT). When no activation occurred, it was assumed that the spermatozoon was deficient; otherwise an oocyte-related factor was suspected. In a subsequent ICSI cycle, AOA was done by ICSI with CaCl₂ followed by a Ca²⁺ ionophore exposure. Fertilization was checked 16–20 h later. Embryo transfer was on day 2 or 3. RESULTS: MOAT showed sperm-related activation deficiency in six globozoospermic patients and two patients with extreme oligoasthenoteratozoospermia. One patient with small sperm acrosomes had a normal activation percentage. In eight other patients, the MOAT revealed a relatively normal activation capacity of the sperm, indicating an oocyte-related defect. After AOA, fertilization rates were 77 and 71% in the sperm- and oocyte-related groups respectively. Five pregnancies were achieved in the globozoospermia group and three in cases of oocyte-related activation failure. CONCLUSIONS: Assisted oocyte activation enables normal fertilization and pregnancy in sperm- and oocyte-related fertilization failure.