Reduced toxicant prototype (RTP) cigarettes with substantially reduced levels of tobacco smoke toxicants have been developed. Evaluation of these prototype cigarettes included measurement of biomarkers of exposure (BoE) to toxicants in smokers switched from conventional cigarettes to the RTPs. A 6-week single-blinded randomised controlled study with occasional clinical confinement was conducted (Trial registration: ISRCTN7215735). All smoking subjects smoked a conventional cigarette for 2-weeks. Control groups continued to smoke the conventional cigarette while test groups switched to one of three RTP designs. Clinical confinement and additional assessments were performed for all smoking groups after 2 and 4-weeks. A non-smoker group provided background levels of BoE. On average, smokers switched to RTPs with reduced machine yields of toxicants had reduced levels of corresponding BoEs. For vapour phase toxicants such as acrolein and 1,3-butadiene reductions of ⩾70% were observed both in smoke chemistry and BoEs. Reductions in particulate phase toxicants such as tobacco-specific nitrosamines, aromatic amines and polyaromatic hydrocarbons depended upon the technologies used, but were in some cases ⩾80% although some increases in other particulate phase toxicants were observed. However, reductions in BoEs demonstrate that it is possible to produce prototype cigarettes that reduce exposure to toxicants in short-term use.