No-till (NT) agriculture has been promoted as one of the optimal management practices that preserves soil and water, and increases soil organic C (SOC) compared with conventional tillage (CT) practices. Information on SOC sequestration in NT systems, however, has been based on measurements from the surface soil (<30 cm) and little is known about the extent of SOC sequestration in NT across the entire 0- to 60-cm soil profile. We conducted a regional study of NT farming to assess the extent of SOC sequestration in the whole soil profile across 12 contrasting but representative soils in the Midwestern United States, each within a Major Land Resource Area (MLRA: 98, 111C, 114B, 122 in Indiana; 111A, 111B, 111D, 124, and 126 in Ohio; and 127 and 147 in Pennsylvania). Soils on gentle terrain were sampled in paired NT and CT fields as well as in an adjacent woodlot in each MLRA. The SOC and N concentrations were greater in the surface 0- to 5-cm soil in NT than CT in MLRA 124. The SOC concentration in CT soil was greater than in NT soil at 10 to 30 cm in MLRAs 98 and 126. The total SOC pool for the whole soil profile did not differ between NT and CT in eight of the 12 MLRAs and the total profile SOC was actually greater under CT in MLRAs 98, 127, and 126, resulting in negative C sequestration rates on conversion from CT to NT in these three MLRAs. This regional study suggests that the entire soil profile must be examined and ecosystem C budget assessed when elucidating SOC sequestration in NT vs. CT fields.