Analysis of bone collagen extracted from human skeletal remains from archaeological sites dating from the Archaic period through Euro-American settlement provides evidence for the introduction of maize into regional subsistence patterns. Stable carbon isotope ratios of samples from both the eastern Ozarks and the Mississippi River alluvial valley indicate that human populations living prior to ca. A.D. 1000 consumed little or no C4 plant material. In populations dating after ca. A.D. 1000, stable carbon isotope ratios indicate that maize represented a significant part of the human diet throughout the region. The change in dietary patterns coincides with a shift in settlement patterns from dispersed hamlets and small villages to civic-ceremonial centers with associated villages, hamlets, and farmsteads. This isotopic evidence indicates that Emergent Mississippian diets did not include substantial quantities of maize or other C4 plant material.