ObjectiveThe aim of this study was to investigate the prevalence of xenobiotic substances, such as caffeine, nicotine and illicit drugs (eg, cannabis and cocaine), in blood samples from first-trimester Danish pregnant women unaware of the screening.DesignA cross-sectional study examined 436 anonymised residual blood samples obtained during 2014 as part of the nationwide prenatal first-trimester screening programme. The samples were analysed by ultra performance liquid chromatography with high-resolution time-of-flight mass spectrometry.SettingAn antenatal clinic in a Danish city with 62 000 inhabitants, where >95% of pregnant women joined the screening programme.Primary and secondary outcome measuresThe prevalence and patterns of caffeine, nicotine, medication and illicit drug intake during the first trimester of pregnancy.ResultsThe prevalence of prescription and over-the-counter drug detection was 17.9%, including acetaminophen (8.9%) and antidepressants (3.0%), of which citalopram (0.9%) was the most frequent. The prevalence of illegal drugs, indicators of smoking (nicotine/cotinine) and caffeine was 0.9%, 9.9%, and 76.4%, respectively. Only 17.4% of women had no substance identified in their sample.ConclusionsThis study emphasises the need for further translational studies investigating lifestyle habits during pregnancy, as well as the underlying molecular mechanisms through which xenobiotic substances may affect placental function and fetal development.