Measurement of the affinity of a protein for a promoter sequence is critical when assessing its potential to regulate transcription. Here we report that the DNA protein crosslinking (DPC) assay can be used to measure affinity, amount and molecular weight of DNA binding proteins to specific and non-specific DNA sequences. By applying a theoretical analysis to evaluate the binding data, it was shown that the affinity constants of two proteins (named DPC80 and DPC107) to the MT3 region of the mouse thymidine kinase promoter were 2 x 10(-9) M, which is 10(4) times higher than to non-specific DNA. Similar affinity constants were found when the purified proteins corresponding to DPC80 and DPC107 instead of nuclear extracts were used to assess the reliability of the DPC assay. A value for crosslinking efficiency was determined as 0.07, however, it is not needed for computation of the DNA-protein affinity, but with it the abundance of a binding protein can be estimated. In summary, the DPC assay is useful for quantifying DNA binding proteins and thereby judging their influence on transcription.