The aim of this study was to evaluate the effect of three concentrations of levonorgestrel (LNG) comparable to the levels found in serum following ingestion of LNG as emergency contraception (EC) on the acrosome reaction (AR) of capacitated and noncapacitated spermatozoa of fertile men. A total of 24 semen samples from three fertile men were evaluated. The spermatozoa were selected by Percoll gradient. Twelve samples were subsequently incubated with human tubal fluid medium supplemented with bovine serum albumin (HTF/BSA) for 20 h under capacitating conditions. The capacitated spermatozoa and the spermatozoa from the remaining 12 samples were exposed to LNG at 1, 10 and 100 ng/mL, to follicular fluid (FF) (20 %v/v) and to HTF medium. The ratio of live to dead spermatozoa was assessed after 1, 2 and 3 h of incubation at 37 degrees C and 5% CO2. After 30 min of exposure to the different LNG concentrations, aliquots were divided into two parts. In the first part, spermatozoa were immediately stained with Hoescht 33258 and fluorescein isothiocyanate-pisum sativum agglutinin (FITC-PSA) in order to assess AR rate and to repeat evaluation of the live-to-dead ratio. After 3 h of incubation, the remaining part of the aliquots were submitted to the same procedures. Each concentration of LNG was then compared with FF and HTF medium as positive and negative controls, respectively. The results showed that in vitro exposure to the three different LNG concentrations did not induce AR. This study failed to show any in vitro effect on AR of LNG concentrations similar to those found in serum following intake of LNG as EC. If this effect exists or if there is any other that influences sperm fertilizing capacity, in vitro experiments are probably not an appropriate way of testing it.72225-8