The DNA-breaking and DNA-breakage-inhibiting activities of 23 steroids (bile acids, steroid hormones, neutral sterols, and oxidized cholesterols) were measured in vitro. No compounds examined broke DNA, but some bile acids such as taurocholic, lithocholic, ursodeoxycholic, chenodeoxycholic, and hyocholic acids inhibited DNA breakage by ascorbic acid. Taurocholic acid had the highest inhibiting activity at concentrations above 10 mmol, but its constituents, taurine and cholic acid, had no activity. On the contrary, glycine was an ihibitor although glycine-conjugated bile acids were not effective. Analysis of the structure–activity relationship of bile acids suggested that the H group but not the OH group in the 12-position of the molecule is required for the DNA-breakage-inhibiting activity of non-conjugated bile acid. Among the conjugated bile acids having the OH group in the 7, 12-positions, taurocholic acid had the DNA-breakage-inhibiting activity, but not glycocholic acid, although glycine, but not taurine, was effective.