The effect of base-pair mismatches on DNA hybridization was investigated by fluorescence polarization to evaluate the specificity of the DNA hybridization technique. A fluorescein-labeled 24-mer DNA probe was used to monitor the course of hybridization, from which the rate of DNA hybridization was obtained. The specificity of the method was considered by comparing the hybridization rate of the fluorescein-labeled probe with complementary oligomers comprising a number of base pair mismatches. In either 0.8 M NaCl or 0.2 M MgCl2, rapidin vitrodetermination of target DNA was possible within 10 minutes if the sample DNA had 3 or less base-pair mismatches. It was shown that this method would be applicable for rapid and specific DNA determination, even in the presence of mismatches.