Abstract: A method for enabling rapid and accurate determination of the number of bacterial cells in a specimen using a PCR method includes the following steps: (1) a first PCR step of carrying out a PCR method using a nucleic acid derived from a specimen as a template and a universal primer pair for amplifying a bacterial 16S rRNA gene to obtain a first amplification product; (2) a second PCR step of carrying out a nested PCR method using a primer pair(s) for amplifying an internal sequence(s) of the sequence of the first amplification product obtained by the first PCR step to obtain a second amplification product; and (3) a bacterial number determination step of obtaining the number of bacterial cells in the specimen based on the amount of the second amplification product obtained in the second PCR step and using calibration data.