Benzene, well known as a ubiquitous environmental pollutant, can lead to increasing risk of cancer, bone marrow failure as well as other serious diseases. Benzene has been classified as carcinogenic to humans with no recommended safe level of exposure. In this study, the influence of time-of-day on benzene metabolism has been tentatively explored in a mouse model based on direct real-time breath analysis by using membrane inlet single photon ionization time of flight mass spectrometry (MI-SPI-TOFMS). The exhaled breath of eight mice was monitored at a time resolution of less than 20 s after intraperitoneal (I.P.) injection of benzene in the morning, afternoon and evening on different days, and two rounds of experiments were carried out in total. The pharmacokinetic parameters such as total exposure AUC0-∞ (h ng/mL), peak level Cmax (ng/mL), time of peak level tmax (h), and terminal half-life t1/2z (h) were calculated and discussed. The values of individual parameter varied greatly among the eight mice, e.g., AUC0-∞ in the morning of the first round of experiment ranged from 10.66 to 162.17 h ng/mL and the mean ± SD was 103.72 ± 99.72 h ng/mL (n = 8). Significant difference has also been observed between two rounds of experiments, implying the damage in the liver caused by the benzene exposure. However, there is no significant difference among the results from the morning, afternoon and evening for each round of the experiment. In our follow-up study, the influence of time-of-day will be further investigated, in which the metabolites of benzene as well as endogenous metabolites will be considered. Copyright © 2017 Elsevier Ltd. All rights reserved.