Acrosin activity was determined using a gelatinolysis technique in 100-microliter semen aliquots of 114 patients (normozoospermia, n = 90; asthenozoospermia, n = 12; oligozoospermia, n = 10; polyzoospermia, n = 2) attending an in vitro fertilization (IVF) program. Halo diameter, halo formation rate, and a calculated acrosin activity index correlated significantly with the IVF rates (P = 0.0054, r = 0.396; P = 0.0009, r = 0.401; and P = 0.0003, r = 0.428, respectively). In cases where the halo diameter was < 10 microns and halo formation rate was < 60%, all patients were subfertile or infertile, that is, they showed poor or no fertilization in vitro, respectively. The assay demonstrated a relatively low sensitivity: 25.7% for halo diameter, 37.1% for halo formation rate, and 25.7% for acrosin activity index, respectively. This might be attributed to other sperm functional aspects, such as disturbed acrosome reaction or impaired zona binding.