The spermatozoa of some patients attending for in-vitro fertilization (IVF) fail to penetrate the zona pellucida in vitro. A test has been devised to identify these cases. It is based on the number of spermatozoa penetrating into the zona pellucida, which were counted after removing spermatozoa bound to the zona surface by vigorous aspiration of each oocyte through a narrow gauge (120 μm) glass pipette. The oocytes were collected from 197 patients undergoing IVF treatment with their own gametes; 79 with no oocytes fertilized and 118 with some oocytes fertilized. Sperm motility, morphology and DNA normality (acridine orange stain) were also measured. The relationships between sperm test results and IVF rate were examined by logistic regression. The proportions of penetrated zonae, normal sperm morphology and normal DNA were the most significant factors related to IVF rate in the whole group. Also, in patients with ≥ 30 spermatozoa bound per zona pellucida or with normal sperm morphology ≥ 30%, the proportion of penetrated zonae and normal DNA were most significant. Oocytes from 42 patients who had zero fertilization and low sperm-zona binding (average, 2.2 spermatozoa/zona pellucida) were re-incubated with normal donor spermatozoa: large numbers of spermatozoa bound (average, 88 spermatozoa/zona pellucida) and each zona was penetrated by at least one spermatozoon. In conclusion, the percentage of zonae penetrated was the variable most significantly correlated with IVF rate. Penetration of the zona was also strongly related to fertilization rates in patients without defects of sperm morphology and sperm-zona binding. In patients where all zonae were penetrated, poor fertilization may be due to sperm morphology and DNA abnormalities. Failure of sperm-zona binding and penetration in vitro in patients with failure of fertilization was mainly due to sperm defects and not oocyte defects