Objective To explore the in vivo exposure levels of cigarette smoking (CS) by measuring the biomarkers nicotine and cotinine.Methods One hundred and sixty male SD rats were divided into 15 cigarette exposure groups (10,20 and 30 nonfilter cigarettes/day,for 2,4,6,8 and 12 weeks) and a control group (without CS exposure).The rats were sacrificed at different time-points.The concentration of plasma nicotine and cotinine were measured by GC-MS/MS.Results The CS-exposed rats displayed decreased locomotor activity,ataxic gait,irregular respiration,nasal noise,and salivation after smoking exposure for 3 weeks.Rats in the CS exposure groups had lower body weight,and the reduction of body weight was time and dose related (P < 0.01).The retention time of nicotine was 7.5 to 8.5 min.The concentration of plasma nicotine in the CS exposure groups was higher than control group (155±56.65) ng/mL.The retention time of cotinine was 11.5 to 12.5 min,the concentrations of plasma cotinine in CS exposure groups were higher than control group (340±41.97) ng/mL,the increase of plasma cotinine in CS groups was time-related (P < 0.05),and the exposure concentration and duration had synergistic effect on the level of plasma cotinine (P < 0.05).Conclusions CS exposure causes structural damages in male rats.The plasma concentration of cotinine can effectively reflect the in vivo exposure levels of cigarette smoking,and well presents a dose-response relationship.