Isotope effects represent perhaps one of the most versatile tools available to investigators interested in the determination of reaction mechanism, particularly in the case of the mechanistic enzymologist. Interpretation of isotope effect data is somewhat more difficult for enzyme reactions, since the chemical or isotope-dependent step(s) is(are) normally not solely rate-limiting as they are for non-enzyme-catalyzed reactions. One can, however, take advantage of rate-limitation by multiple steps in an enzyme-catalyzed reaction to obtain information on a number of aspects of mechanism. In this paper, simple theory for the application of isotope effects to reaction mechanism is developed, and applied to organic reactions and those catalyzed by enzymes. Techniques used to measure isotope effects depend somewhat on the isotope used, that is radioisotopevs.stable isotope, or hydrogen isotopevs.heavier atoms. Techniques to be discussed include competitive and noncompetitive (or internal discrimination) measurements. In enzymecatalyzed reactions, information can be obtained on the order of addition of reactants and release of products, and this will be illustrated using the 6-phosphogluconate and alcohol dehydrogenase reactions. The use of multiple isotope effects can be used to distinguish between stepwise and concerted reactions, and this will be illustrated with the formate and glucose 6-phosphate dehydrogenase and malic enzyme reactions.