G2 banding of human peripheral blood cultures with actinomycin D and tetracycline produced chromatid breakage in lightly stained Giemsa bands, and the number of such breaks tended to increase with fixation time. Chromatid breakage due to 3H-thymidine incorporation into DNA was also localized in light bands, but the number of these breaks did not increase with fixation time. These findings suggest that modification of chromosomal protein as a result of exposure to AMD or other chemicals following DNA synthesis can result in fixative-dependent chromatid breakage of susceptible chromosomal regions, unlike breakage due to 3H-thymidine which primarily affects DNA and is not affected by fixation. Thus, chromatid breakage observed in short-term lymphocyte cultures is not necessarily evidence of mutagenicity involving DNA, but rather may be due to toxic effects on synthesis or attachment of chromosomal proteins.