The reaction of calicheamicin gamma(1) (1) with glutathione (GSH) has been studied in the presence of double-stranded DNA and is shown to produce initially all four products arising from S-S bond exchange between the calicheamicin gamma(1) trisulfide group and the thiol function of glutathione. The calicheamicin-glutathione disulfide 6 is formed as the major product of the reaction, while the dihydrothiophene derivative 3, the thiosulfenic acid derivative 4, and the calicheamicin-glutathione trisulfide 5 are formed as comparatively minor products. The product distribution is highly time dependent for products 4-6 undergo further transformation, ultimately producing 3. The rates of reaction of products 4 and 5 with GSH are roughly comparable to that of 1, while the calicheamicin-giutathione disulfide 6 forms 3 at a rate approximately 2 orders of magnitude slower than the rate of formation of 3 from 1. The kinetics of cleavage of double-stranded DNA by 1 and GSH, determined by polyacrylamide gel electrophoretic (PAGE) analysis is found to parallel the kinetics of formation of 3 and is characterized by a two-stage process. Both stages of DNA cleavage proceed with identical sequence specificity. The rates of DNA cleavage by 1 and 6 respond in different fashion to variations in the concentration of DNA. The rate of DNA cleavage by 1 is essentially independent of the concentration of DNA, while the rate of DNA cleavage by 6 is inversely proportional to the concentration of DNA. The data support the hypothesis that 1 undergoes thiol activation as a DNA-bound species, while 6 is activated free in solution. These findings suggest that, under physiologically relevant conditions, the major DNA-damage pathway arising from the reaction of 1 and GSH involves the following sequence : 1 binds to double-stranded DNA; DNA-bound 1 reacts with GSH to form 6, DNA-bound 6 dissociates and reacts with free GSH to form A and then 2; the product(s) of the latter reaction (likely 2) bind to DNA; DNA-bound 2 rearranges to the biradical B, which then abstracts hydrogen atoms from the ribose backbone of DNA. New and existing data pertaining to the potential role of DNA as a catalyst in the thiol activation reaction and to the potential participation of the carbohydrate amino group of 1 in that reaction is evaluated. It is determined that while there is evidence to support the hypothesis that the carbohydrate amino group of 1 participates in the thiol activation of 1 in organic solvents, no conclusions may be drawn at this time concerning its role in the corresponding reaction in water in the presence of DNA. Similarly, it is concluded that there is presently insufficient data to determine if DNA functions as a formal catalyst for the thiol activation of 1 in water.