Unlike their parent strains, zidovudine-resistant derivatives of Escherichia coli KL16 and Salmonella typhimurium NCTC 5710 were found to be incapable of incorporating radiolabelled thymidine into their chromosomal DNA. Since incorporation was still prevented in the presence of EDTA, resistance to zidovudine was not associated with a permeability barrier, but appeared to result from the loss of thymidine kinase activity, required for the phosphorylation of zidovudine. Pseudomonas aeruginosa, which is intrinsically zidovudine-resistant, was also shown to be incapable of incorporating thymidine into its DNA, but Staphylococcus epidermidis SK360 and Staph. aureus E3T, which are also intrinsically zidovudine-resistant, possessed thymidine kinase activity. This suggests that two distinct mechanisms of resistance to zidovudine exist in bacteria. Zidovudine resistance did not appear to confer resistance to other antibacterial agents.