The usefulness of sub-zonal injection (SZI) for the treatment of severe male factor infertility has been restricted by low and unpredictable fertilization rates and the high risk of polyspermy after the injection of multiple spermatozoa. In this prospective study, we have evaluated whether sperm morphology and the percentage of acrosome-reacted spermatozoa at the time of injection can be used to predict SZI fertilization outcomes. Populations of motile spermatozoa equivalent to those injected were collected from the medium/oil interface immediately after SZI of each cohort of oocytes. Morphology was assessed using the World Health Organization 1987 criteria and the acrosomal status of spermatozoa was determined after staining with rhodamine-conjugated Pisum sativum agglutinin. A fertilization index (FI) was calculated to express the actual fertilizing potential of the spermatozoa injected. In all, 67 patients underwent 72 SZI cycles. The overall fertilization and polyspermy rates were 36 and 47% respectively, and a clinical pregnancy rate per transfer of 22% was achieved. Linear regression analysis demonstrated a statistically significant relationship between morphology and the FI (r = 0.506, P < 0.0001). Patients with < or = 10% normal morphology always had a FI < or = 10%, and this was reflected by low fertilization and polyspermy rates and the high number (32%) of cycles with complete failure of fertilization in this group. In patients with > 10% normal morphology, there were two patterns: low (< or = 10% FI) or high (> or = 10% FI) fertility. This was evident in the fertilization (23 and 85%, respectively), and polyspermy (25 and 68%, respectively) rates of these two patient sub-groups.(ABSTRACT TRUNCATED AT 250 WORDS)