The effects of NaCl concentration and temperature on the rate of hybridization of complementary single-stranded DNA (24-mers) were investigated. The single label of fluorescein was used for the probe DNA. The time courses of fluorescence polarization for the probe DNA were monitored. It was shown that detection of a specific DNA sequence (24-mer) was possible in less than 10 min using fluorescence polarization under the optimized conditions of 0.8 M NaCl at 46°C in TE buffer. The effects of base-pair mismatches on DNA hybridization in the presence of NaCl or MgCl2 were also investigated, and the specificity was considered by comparing the hybridization rate of the fluorescein-labeled probe. Determination of a specific DNA sequence was also possible in TE buffer containing 0.2 M MgCl2. Moreover, in the presence of 0.2 M MgCl2, there were no undesirable effects on hybridization and the presence of a single base pair mismatch could be identified. Rapid and specific determination of the DNA of enterohemorrhagic Escherichia coli, methicillin resistant Staphylococcus aureus and Legionella pneumophila, which had been multiplied by the asymmetric PCR, was performed under the optimized conditions for hybridization. It was confirmed that the conditions were also applicable to the hybridization between the probes and the amplified products of the actual bacterial genes. The combination of fluorescence polarization with the asymmetric PCR was quite effective. Moreover, the nested and asymmetric PCR product of bacterial gene could be detected effectively. The DNA detection method could also be used even if the specificity of the DNA amplification was not perfect and some unexpected bands were mixed with the target band during electrophoresis.