A steady state kinetic study of Escherichia coli DNA polymerase I has been carried out using poly[d(A-T)] as the template-primer substrate. The results of substrate saturation and product inhibition kinetic studies suggest an altered Ordered Bi Bi mechanism for the enzyme. The Michaelis constants for polymer, d-atp, and dTTP are 5 nM (3'-OH ends), 1 muM, and 2 muM, respectively. The apparent equilibrium constant for the reaction, Keq equals [PPi]/[dNTP], was estimated as greater than or equal to 500. No quaternary complex of enzyme, template, and both deoxynucleoside triphosphates was detected. Single turnover experiments at 4 degrees indicated that the enzyme functions non-processively under the specified conditions, that is, dissociates after each catalytic step. The results at higher temperature were consistent with dissociation within 30 steps. Furthermore, at 4 degrees a burst of incorporation stoichiometric with the amount of enzyme was observed upon initiation of the reaction, indicating that the rate-limiting step in the steady state occurs after phosphodiester bond formation. There is a linear Arrhenius dependence of the initial reaction on temperature in the range 4-40 degrees, with an apparent Ea equals 17 kcal/mol. The rate equations appropriate for template-dependent polymerases which dissociate after each catalytic step have been derived.