The present study was conducted to evaluate the effect of in vitro treatment of ejaculated ram spermatozoa with different levels of theophylline at various incubation times on motility, hyperactivity (HA) and acrosome reaction (AR). Semen were collected from Barki rams (n=40 ejaculates) using artificial vagina twice a week. Collected semen were pooled and subjected to swim up technique in modified sperm Tyrode's albumin lactate pyruvate (S-TALP) medium supplemented with different levels of theophylline (0.01, 0.02, 0.03, 0.04 and 0.05 mg/ml) at different incubation times (0, 1,2,3 and 4 hours). The following parameters were examined: motility, hyperactivity (HA) and acrosome reaction (AR %). The results revealed that highest motility was noticed in spermatozoa treated with 0.05 mg/ml theophylline (82.53±1.34%) immediately after dilution. While, addition of 0.01 mg/ml theophylline resulted in a significant (P<0.05) increase in HA% than those obtained in control (untreated) after 3 hours of incubation (5.04±0.26 vs.0.8±0.2). On other hand, a significant (P<0.05) increase in total AR% was concomitant to the increase in the concentration of theophylline with maximum achieved at 0.05 mg/ml at 3- and 4-hours post incubation (49.2±1.8; 51.2±2.5, respectively). In conclusion, under our experimental conditions, treatment of ram spermatozoa with 0.05 mg/ml of theophylline for 3-4 hours was considered the best concentration of theophylline to be used for in vitro induction of acrosome reaction.