The prediction of male fertility is an important aspect of livestock production. Until now, a routine semen analysis including sperm motility and morphology is widely used as a fundamental indicator of male fertility. However, it does not provide precise information about the fertilizing capacity of sperm, because the evaluation standard frequently fails to be predictive of male infertility, and fertilization sometimes occurs even in the presence of abnormal semen analysis. In addition, there is an event of semen with low fertility, though there is little apparent overall difference on a routine semen analysis, and this is now becoming a serious problem in animal production. Therefore, we developed a simple procedure for evaluating the fertilizing ability of boar sperm based on the properties of acridine orange (AO), which fluoresces green when bound to intact, double-stranded DNA and yellow or red when bound to denatured DNA (single-stranded). Based on conception rates after natural mating or artificial insemination, the boar semen samples assigned to fertile (n=5) and subfertile (n=4) groups were studied. Semen quality of both groups was within the normal ranges in a routine semen analysis. When AO staining after low pH-induced denaturation was carried out on sperm smears, in fertile group the percentage of yellow or red-fluorescing sperm heads remained low (3%), indicating that the majority of sperm heads contained a chromatin resistant to denaturation. In subfertile group a high percentage of yellow or red-fluorescing sperm heads was found (37%), revealing the presence of cells whose chromatin was sensitive to denaturation. In addition, AO data was well reflected in in vitro fertilization rates, which were significantly higher in fertile group than subfertile group (72% vs. 4%; p<0.01). From these findings, therefore, it is possible that increased susceptibility to denaturation causes decreased structural stability of sperm nuclear chromatin, suggesting that the AO staining method could be a useful tool for diagnosing subfertile boars.