5,6-Dihydrothymidine (dDHT) is a derivative thymidine formed during gamma-irradiation. This paper demonstrates the conditions under which dDHT is formed in solutions of DNA and that dDHT is produced in the DNA of HeLa cells during gamma-irradiation. The product of dDHT by gamma-irradiation of either thymidine or DNA has been quantitated by a sensitive and specific high-pressure liquid chromatography method. dDHT is a major product of the anoxic irradiation of thymidine (G value 0.5) but is produced in substantially smaller amounts in DNA irradiated under the same conditions (G value 0.026). The presence of oxygen reduces the yield of dDHT by at least 25-fold for both irradiation substrates. In HeLa cells, 60Co irradiation under anoxia produces (6.2 +/- 0.2) X 10(-8) mol of the R isomer of dDHT per mole of cell deoxynucleotide per gray (G value 0.11). gamma-Irradiation of thymidine produces equal quantities of the R and S stereoisomers of dDHT. Irradiation of DNA produces significantly more (69%) (R)- than (S)-dDHT. DNA isolated from cultured human cells following gamma-irradiation also contains more of the R than the S form of dDHT. The conformation of double-stranded DNA favors a stereospecific production of the R isomer. Among products of gamma-irradiation of DNA, dDHT is unique in its strict requirement for anoxia during irradiation and the preferential production of a particular stereoisomer.