We investigated the in vitro effects of arsenic trioxide on cell growth, cell cycle regulation, and apoptosis in As4.1 juxtaglomerular cells. Arsenic trioxide inhibited the growth of As4.1 cells with an IC₅₀ of approximately 5 µM. Arsenic trioxide induced S phase arrest of the cell cycle, and very efficiently stimulated apoptosis in As4.1 cells, as evidenced by flow cytometric detection of sub-G₁ DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial transmembrane potential (_m), a decrease in Bcl-2, the activation of caspase-3 and cleavage of PARP. However, all of the caspase inhibitors tested in this experiment failed to rescue As4.1 cells from arsenic trioxide-induced cell death in view of sub-G₁ cells and annexin V positive staining cells. However, caspase-8 inhibitor (Z-IETD-FMK) noticeably decreased the loss of mitochondrial membrane potential (_m) in arsenic trioxide-treated cells. When we examined the changes of the ROS, H₂O₂ or O₂^·-, in arsenic trioxide-treated cells, H₂O₂ was significantly decreased and O₂^·- was increased. In addition, we detected a decreased GSH content in arsenic trioxide-treated cells. Taken together, we have demonstrated that arsenic trioxide as a ROS generator potently inhibited the growth of As4.1 JG cells through S phase arrest of the cell cycle and caspase-independent apoptosis.