The present study shows the use of confocal autofluorescence spectroscopy coupled with the time-resolved fluorescence decay analysis to measure changes in FAD/NAD[P]H and free/bound NAD[P]H in HepG(2) cells at 0.5, 1.5, 3 and 4.5h after exposure to cadmium chloride (Cd). These changes were compared to changes in GSSG/GSH and production of reactive oxygen radicals (ROS) production. The results demonstrated that both FAD/NAD[P]H and GSSG/GSH increased significantly upon exposure to Cd. The change in GSSG/GSH occurred as early as 1.5h after treatment while the change in FAD/NAD[P]H did not occur until 3h after exposure. Production of ROS was also increased at 1.5h. The ratio of free/bound NAD[P]H was studied. It was demonstrated that free/bound NAD[P]H increased significantly as early as 0.5h and remained elevated until 4.5h after treatment with Cd. The present study provides novel data to show that changes in NAD[P]H metabolism precedes the increase in ROS production and cellular oxidative stress (increase GSSG/GSH, FAD/NAD[P]H). It is suggested that Cd causes a release of NAD[P]H, an important cofactor for electron transfer, from its normal protein binding sites. This may result in a disruption of the activity of the enzyme and proteins, and may lead to the subsequent toxic events.