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Nicotine is rapidly and extensively metabolized in humans and negatively impacts the developing fetus. The concentrations of nicotine, cotinine, trans-3?-hydroxycotinine (hydroxycotinine), and norcotinine in pregnant smokers' oral fluid were evaluated to determine usefulness as biomarkers of cigarette smoking. Sixteen participants were divided into two groups: eight light smokers (LS) who smoked ? 10 cigarettes/day and eight heavy smokers (HS) who smoked ? 20 cigarettes/day. Oral fluid specimens (n = 415) were collected throughout pregnancy and analyzed with solid-phase extraction followed by gas chromatography–mass spectrometry-electron impact selected ion monitoring. Median concentrations of nicotine, cotinine, and hydroxycotinine in oral fluid of LS ranged from 241.1 to 622.0, 80.6 to 387.5, and 14.4 to 117.7 ng/mL and for HS 146.5–1372.2, 66.0–245.8, and 38.3–184.4 ng/mL, respectively. Salivary cotinine and hydroxycotinine concentrations were significantly correlated in LS (r = 0.55, p < 0.01) and HS (r = 0.74, p < 0.01). Ratios of hydroxycotinine/cotinine in oral fluid from pregnant women averaged 0.30 ± 0.18 (range, 0.07–1.05) for LS and 0.68 ± 0.25 (range, 0.29–1.83) for HS. Based on these preliminary data, the best ratio to differentiate light from heavy pregnant smokers was 0.41. Salivary hydroxycotinine and cotinine concentrations are both good biomarkers of cigarette smoking. Determining the hydroxycotinine/cotinine ratio may differentiate light from heavy tobacco use and help predict increased fetal tobacco exposure.