The ultraviolet (UV) absorbance ratio of 260/280 nm has been used as an indicator of DNA purity. However, the A260/A280 ratio may be beyond the normal range (1.8-1.9) due to physicochemical alterations produced by pH and temperature, and carcinogenic chemical modification. When the pH of the DNA solution buffer increased from 3 to 11, the A260/A280 ratio changed significantly from 1.5 to 2.2 in mixtures of DNA bases [A:T:C:G = 28.5:28.5: 21.5:21.5, i.e., (A + T)/(all four bases) = 57%, expressed as mole percent], of deoxyribonucleosides (adenosine:thymidine:cytidine:guanosine= 28.5:28.5:21.5:21.5, as mole percent), or of deoxyribonucleotides (dAMP:dTMP:dGMP:dCMP = 28.5:28.5:21.5:21.5, as mole percent) examined. The A260/A280 ratio increased with RNA contamination and exceeded 1.9 when RNA concentration was >30%, as mole percent. In contrast, the A260/A280 ratio was linearly reduced by increasing the protein concentration. Phenol (>0.02%) contamination also reduced the A260/A280 ratio to below 1.8. Benzo[a]pyrene diol epoxide (BPDE), a reactive carcinogen metabolite of benzo[a]pyrene (BaP), decreased the A260/A280 ratio correlated with the degree to which it modified the DNA. These results suggest that the UV A260/A280 ratio is significantly affected by pH and the presence of contaminating species of macromolecules and chemicals.