The aim of this study was to determine the effects of in vitro treatment of freshly ejaculated ram spermatozoa with different concentrations of penicillamine, hypotaurine, and epinephrine (PHE) at different incubation times on motility, hyperactivity (HA) and acrosome reaction (AR). Freshly ejaculated spermatozoa collected from three rams were pooled and then subjected to swim up technique in modified sperm Tyrode’s albumin lactate pyruvate (S-TALP) medium supplemented with different concentrations of PHE (0, 10, 20, 30, 40, 50, 75, and 100 mm). Following incubation (0, 1, 2, 3 and 4 h), sperm motility and hyperactivity were examined under the phase contrast microscope and acrosome reaction was detected by staining of the spermatozoa with silver nitrate. Results showed that treatment of spermatozoa with high concentrations of PHE (30, 40, 50, 75, and 100 mm) significantly increased the motility when compared with the control immediately after dilution (82.0, 82.0, 81.0, 82.0, 82.0 v.76.0%, respectively).This increase existed for the first and second hours of incubation. However, when the incubation time was increased for more than 2 h total motility significantly decreased (P < 0.05, ANOVA) as compared with the control. The same trend was observed in hyperactive motility. Treatment of spermatozoa with 50 and 75 mm of PHE for 1 h significantly increased the percentage of sperm with incomplete AR (20.0 and 27.0% respectively). At 4 h incubation, (49.0%) of spermatozoa treated with 75 mm PHE had undergone complete AR. Furthermore, the maximum value of total acrosome reaction (73.0%) was achieved at 4 h post incubation after addition of 75 mm PHE to ram sperm. In conclusion, under our experimental conditions, 75 mm PHE for 4 h was considered the best concentration of PHE for treatment of ejaculated ram spermatozoa for in vitro induction of acrosome reaction.