We have used coupled carbon and nitrogen isotope measurements together with molecular level isotope analysis of biomarker molecules to assess environmental changes across two nonmarine Cretaceous-Tertiary (K-T) boundary successions in the Western Interior of North America. Measurement of the carbon isotope compositions of individual molecules greatly increases the usefulness of biomarkers as indicators of specific sources.The terrestrial organic matter carbon isotope record apparently reflects the 2‰-3‰ isotope excursion observed in the marine foraminiferal record, and the nitrogen isotope record contains a small positive excursion immediately above the boundary. Biomarker analysis indicates that the two major organic matter inputs were higher plants and algae. However, the relative contributions of these two sources vary above the K-T boundary toward a greater input from algae and bacteria. Biomarker and isotopic evidence for the presence of a methane-oxidizing bacterial community is also recorded immediately above the boundary, suggesting the onset of partially anoxic conditions in freshwater ecosystems.