Bovine pituitary extract (BPE) is routinely used as a mitogenic supplement in serum-free growth medium. In addition to its mitogenic activity, BPE contains a variety of growth factors and hormones with reported antioxidant activity. This study examines the antioxidant potential of BPE in nontumorigenic human prostate epithelial cells (RWPE-1). Treatment of RWPE-1 cells with BPE (50 μg/ml) provided significant protection against H₂O₂-induced cell death, deoxyribonucleic acid fragmentation, protein oxidation, and membrane damage. Treatment with heat (71° C, 10 min) and proteolytic enzymes reduced the antioxidant activity of BPE, suggesting that proteins present in BPE may be responsible for the antioxidant activity. Residual catalase activity present in BPE was responsible for a portion (30%) of the antioxidant activity. Interestingly, RWPE-1 cells treated with BPE and H₂O₂ rapidly accumulated intracellular reactive oxygen species (ROS) to a greater extent than cells receiving only H₂O₂. Pretreatment of RWPE-1 cells with tyrosine kinase inhibitors (genistein, tyrphostin 47, and AG-1296) before the addition of H₂O₂ diminished BPE protection against H₂O₂-induced cell death, whereas treatment with purified mitogens commonly found in BPE, growth hormone and basic fibroblast growth factor, did not protect against oxidative damage. Taken together, these data suggest that BPE contains proteins or protein complexes with remarkable antioxidant activity. These yet unidentified compounds appear to confer protection against H₂O₂-induced cell death by tyrosine kinase–dependent pathways that increase intracellular ROS generation. The antioxidant activity of BPE may represent a confounding variable when studying oxidative stress in cells maintained in BPE-supplemented serum-free medium.