We have recently reported, in a small cohort of subjects, that acrosome reaction (AR) and intracellular free calcium ([Ca²⁺]_i) increase in response to progesterone were significantly correlated with in-vitro fertilization (IVF) rate. In the present study we extended these results to 90 subjects undergoing IVF. We confirm that both parameters were highly significantly correlated with the fertilization rate (P<0.001). In particular, significantly lower responses to progesterone were detected in subjects with a fertilization rate <50%, further enlightening the functional significance of sperm responsiveness to progesterone with respect to the process of fertilization. Moreover, we report here that both tests are highly discriminant of fertilization success, with positive predictive values >90% for [Ca²⁺]_i values which increase by >1.2-fold and AR inducibility >7% (cutoff values). Conversely, AR following challenge with the calcium ionophore A23187 was less significantly correlated with the percentage fertilization rate (P<0.05), and showed lower predictive values than response to progesterone. All these tests ([Ca²⁺]_i increase in response to progesterone, AR in response to progesterone and to A23187) appear highly sensitive and moderately specific The positive predictive value may rise to >95% when the combination of two tests ([Ca²⁺]_i and inducibility of AR in response to progesterone) is considered. No correlation with fertilization rate has been found for spontaneous AR or basal [Ca²⁺]_i. In conclusion, we propose that assessment of human sperm responsiveness to progesterone may be clinically useful in predicting fertilizing ability in vitro.