Dust Cave (1Lu496) is a habitation site in a karstic vestibule in the middle Tennessee River Valley of Northern Alabama. The cave, periodically occupied over 7,000 years, contains well-preserved bone and botanical materials and exhibits microstratigraphy and intact occupation surfaces. The chronostratigraphic framework for Dust Cave is based on 43 14C dates, temporally diagnostic artifacts, and detailed geoarchaeological analysis. In a broad sense, five cultural components are defined and designated: Quad/Beaver Lake/Dalton (10,650–9200 cal B.C.), Early Side-Notched (10,000–9000 cal B.C.), Kirk Stemmed (8200–5800 cal B.C.), Eva/Morrow Mountain (6400 to 4000 cal B.C.), and Benton (4500–3600 cal B.C.). Microstratigraphic and artifact analyses indicate that the primary differences in the deposits over time relate to intensity of activity and spatial organization with regard to changing conditions in the cave, not to the types of activities. Geomorphic transformations influenced the timing of occupation at Dust Cave, especially the initial occupation. The chronostratigraphy provides a framework for assessing the stratigraphic separation of Dalton and Early Side-Notched materials, the shift in technology from blades to bifacial tools, and the context of detailed flora and fauna evidence. These remains provide unique insights into forager adaptations in the Midsouth from the end of the Pleistocene through the first half of the Holocene.