The kinetic mechanism and the structural bases of the fidelity of DNA polymerases are still highly controversial. Here we report the use of three probes in the stopped-flow studies of Pol beta to obtain new, direct evidence for our previous interpretations: (a) Increasing the viscosity of the reaction buffer by sucrose or glycerol is expected to slow down the conformational change differentially, and it was shown to slow down the first (fast) fluorescence transition selectively. (b) Use of dNTPalphaS in place of dNTP is expected to slow down the chemical step preferentially, and it was shown to slow down the second (slow) fluorescence transition selectively. (c) The substitution-inert Rh(III)dNTP was used to show for the first time that the slow fluorescence change occurs after mixing of Pol beta.DNA.Rh(III)dNTP with Mg(II). These results, along with crystal structures, suggest that the subdomain-closing conformational change occurs before binding of the catalytic Mg(II) while the rate-limiting step occurs after binding of the catalytic Mg(II). These results provide new evidence to the mechanism we suggested previously, but do not support the results of three recent papers of computational studies. The results were further supported by a "sequential mixing" stopped-flow experiment that used no analogues, and thus ruled out the possibility that the discrepancy between experimental and computational results is due to the use of analogues. The methodologies can be used to examine other DNA polymerases to answer whether the properties of Pol beta are exceptional or general.