In order to explore the early apoptotic signal messengers and to search the apoptotic pathway, the morphological and functional changes of mitochondria were examined, and nitric oxide (NO) and calcium ions (Ca2+) were measured in the course of apoptosis in esophageal carcinoma cells induced by As2O3. The esophageal carcinoma cell line SHEEC1, established by HPV in synergy with TPA in our laboratory, were cultured with serum-free medium in a culture flask, 24-well plate and small culture chambers, and added with As2O3 at 1, 3, 5 micromol/l. After 0, 2, 4, 8, 12, 24 h of drug adding the NO were measured from extracellular cultured medium and the SHEEC1 cells were collected from flasks for electron microscopic examination. Fluorescent intensity (FI) of rhodamine 123 (Rho123) labeled cells was detected using laser confocal scanning microscope (LCSM) for evaluation of mitochondrial membrane potential. Intracellular Ca2+ of cells in small culture chambers labeled with Fluo-3 dye were measured using LCSM over time. After adding As2O3, SHEEC1 cells revealed characteristic morphological and functional changes of mitochondria such as hyperplasia, swelling and disruption, accompanying decrease of transmembrane potential (FI of Rho123 decreased). The Ca2+ level increased at once after adding As2O3 and the NO concentration increased step by step till 24 h, then apoptotic morphology of cells occurred. The results suggest that by inducement of As2O3 increasing Ca2+ and NO, the apoptotic signal messengers, will initiate the mitochondria-dependent apoptotic pathway.