In order to use sensitive molecular-level biomarkers for the evaluation of environmental risks, it is necessary to establish a quantitative dose-response relationship. Passive dosing is regarded as a promising new technique for maintaining a constant exposure condition of hydrophobic chemicals in the assay medium. The main goals of the present study were (1) to quantitatively compare gene expression results obtained using the passive dosing method and the conventional spiking method and (2) to investigate changes in gene expression with respect to the free concentration and exposure duration using passive dosing. Chlorpyrifos (CP), which is oxidized by the cytochrome P450 monooxygenases, was selected as a model chemical, and the expression of cytochrome P450 subfamily protein 35A gene series (cyp-35a1-5) was analyzed by quantitative real-time PCR on soil nematode Caenorhabditis elegans. Whereas the free concentration of CP rapidly decreased and the expression of cyp genes varied with the volume of exposure medium and the test duration when the spiking method was used, the free concentration in the assay medium was stable throughout the experiment when the passive dosing method was used. In addition, the level of gene expression increased with exposure time up to 8 h and with increasing CP concentration. The observed increased gene expression could be explained by increasing body residue concentration of CP with exposure time. In conclusion, quantitative dose-response relationships for gene expression biomarkers could be obtained for highly hydrophobic chemicals when the constant exposure condition is provided and the free concentration is used as the dose-metric.