24-norcholestanes have been shown to be useful biomarkers to assess the age of sediments and petroleum, but until now, the biological sources of their precursors, i.e., 24-norsterols, were unclear. We have unambiguously identified relatively high concentrations of 24- norcholesta-5,22-dien-3β-ol in the diatom Thalassiosira aff. antarctica (6%–10% of total sterols) and, in much lower concentrations, in the dinoflagellate Gymnodinium simplex (0.2% of total sterols). These identifications and other reports of 24-norsterols in dinoflagellates suggest that both diatom and dinoflagellate species are major sources for 24-norcholestanes in sediments and petroleum. The evolutionary history of these organisms suggests that observed increases of 24-norcholestane concentration in the Jurassic and the Cretaceous are related to dinoflagellate expansion, whereas an increase in the Oligocene-Miocene is likely caused by diatom expansion. Our results also explain the biogeographical distribution of 24-norcholestanes, i.e., high concentrations at high (paleo)latitudes are likely caused by diatoms, while low concentrations at lower (paleo)latitudes are likely caused by dinoflagellates.