We investigated several factors, such as temperature, current intensity (i), time (t) and the product (mA min mm(-2), viz., C mm(-2)) of i and t, etc., that obviously affect the moving neutralization reaction boundary method (MNRBM). The results manifest that the temperature and the product ti have a strong influence on the movement rate of the boundary. The data prove that about 0.6 C mm(-2) (being equivalent to 10 mA min mm(-2)) is a critical point. If the product ti is lower than the critical point, a good quantitative agreement exists between the observed and theoretical values, but if it is higher than the critical point, the agreements are poor. The optimized experimental conditions are: (1) 18-20 degrees C room temperature, (2) 0.6-0.8 mA mm(-2), (3) less than 10 mA min mm(-2), (4) 1% agarose gel, (5) daily prepared solution and gel containing NaOH. The optimized MNRBM is of benefit for the studies on MNRB itself, isoelectric focusing and capillary zone electrophoresis as will be partially shown in this paper.