Consumption of roll-your-own (RYO) tobacco is rising, but little is known about its in vivo delivery of toxins relative to factory-made (FM) cigarettes. To start to address this issue, this study compared the concentrations of metabolites of recognized human carcinogens in smokers of RYO tobacco and FM cigarettes. We opportunistically recruited 127 FM and 28 RYO cigarette smokers in central London and collected saliva and urine samples. Saliva samples were assayed for cotinine while urinary samples were assayed for 1-hydroxypyrene (1-HOP) and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), metabolic markers of polycyclic aromatic hydrocarbons and tobacco-specific N-nitrosamines, respectively. Data on socio-demographic, anthropometric and puffing characteristics were also obtained. Both unadjusted and adjusted analyses (controlling for age, sex, body mass index, puff flow, puff duration and cotinine) showed no difference in metabolic markers between RYO and FM cigarette smokers. However, significant main effects for cotinine levels and sex were observed in adjusted analyses. Greater levels of cotinine were associated with a greater concentration of both 1-HOP (B = 0.002, P = 0.037) and NNAL (B = 0.002, P < 0.001). In addition, women had significantly greater concentrations of urinary 1-HOP (B = 0.679, P = 0.004) and total NNAL metabolites (B = 0.117, P = 0.024) than men, irrespective of the type of cigarettes smoked. More research is now needed to confirm these findings and gender-specific effects in a larger, representative sample. However, results do not support the common belief that RYO cigarettes are less harmful than manufactured cigarettes.