To continue our mutagenesis study of the polymerase active site of the Klenow fragment of DNA polymerase I, we have characterized new mutants with substitutions at Asp705, Glu710, and Glu883, and have investigated further the mutations in residues Arg668, Gln849, and Asp882, which showed strong Kcat effects in our previous study. To determine which step of the reaction is rate-limiting for each mutant protein, we measured the effect on the reaction rate of an alpha-thio-substituted dNTP. One group of mutants showed a substantial sulfur elemental effect, while a second group, like wild-type Klenow fragment, was unaffected by the phosphorothioate substitution. Consistent with earlier data, these results imply that, between formation of the enzyme-DNA-dNTP ternary complex and completion of phosphodiester bond formation, there are two kinetically distinct steps, only one of which is sensitive to sulfur substitution. The rather complex elemental effect data obtained with the mutant derivatives of Klenow fragment are more consistent with the elemental effect being the result of a steric clash in one of the transition states than with explanations based on electronegativity differences between sulfur and oxygen. The data suggest that the side chain of Asp882 is involved in the proposed steric clash, and that Gln849 and Glu883 participate in the sulfur-sensitive step of the reaction. Based on our results, and comparisons with other phosphoryl transfer enzymes, possible mechanisms for the polymerase reaction are discussed.