Background: The U.S. FDA has the authority to reduce cigarette nicotine content if found to benefit publichealth. Reduced nicotine content (RNC) cigarette use does not appear to increase harm exposure, but studies have not rigorously assessed smoking behavior or used a comprehensive panel of biomarkers. This study examined the effects of progressively decreasing RNC cigarettes on smoking behaviors, biomarkers of exposure, and subjective ratings. Methods: One hundred and fifty-eight daily, non-treatment-seeking smokers participated in a 35-day randomized, unblinded, parallel study. After a 5-day baseline period, participants were randomly assigned to an experimental group (n = 80) that smoked progressively decreasing RNC cigarettes during three 10-day periods, or control group (n = 78) that smoked their own brand throughout the study. Results: Daily cigarette consumption significantly increased for the intermediate RNCs (P's < 0.001) but approached baseline rate for the lowest RNC (P = 0.686); in contrast, puffing behavior significantly decreased at intermediate levels and increased for the lowest RNC (P's < 0.001). Cotinine and NNAL significantly decreased by RNC period (P's = 0.001-0.02), whereas CO boost initially increased (P's = 0.001-0.005). 1-HOP did not change by period (P = 0.109). Conclusions: Smoking behaviors changed by RNC period via CPD and puffing behavior. Biomarkers of exposure generally decreased with nicotine content. Impact: Findings suggest that RNC use does not ubiquitously reduce smoking behaviors or biomarkers, yet the lowest RNC level tested may reduce harm exposure. This emphasizes the importance of using multiple behavioral and biologic measures to address the impact of RNC cigarette smoking.