The reaction kinetics of copper interaction with dissolved organic matter (DOM) in water were studied in order to determine the effect of equilibration period on the toxicity of copper to aquatic organisms. The changes in physical and chemical forms of the copper during four reaction times were examined in four completely mixed reactors in series; the bioavailability of the copper as a function of these new forms was then determined with a flow-through bioassay system, using Ceriodaphnia dubia as a test organism. This study showed that the toxicity of copper to C. dubia decreased with increasing copper-DOM reaction time, which demonstrated that the copper reaction rate with dissolved organic components in the test water was slow. The toxicity of copper to C. dubia was closely related to the measured free-copper concentration (Cu2+) rather than to the total copper concentrations, a fact that supports the free ion activity model. We found that the LC50 of copper for C. dubia increased (i.e., toxicity decreased) linearly with increasing total available binding sites. Although a similar trend was observed in both natural DOM and commercial humic acid, our results indicated that for a given copper-organic carbon ratio, copper binds more strongly to humic acid than to the natural DOM. This difference may be attributed to the greater copper binding affinity of humic acid (greater than that of other metal-binding organic fractions present in DOM, i.e., fulvic acid).