δ13C values of 142 samples from the Manitoulin Formation and subjacent strata collected from 14 exposures and two drill-cores on Manitoulin Island, Bruce Peninsula, and the region south of Georgian Bay suggest that the Manitoulin Formation is latest Ordovician (Hirnantian) rather than earliest Silurian in age. A δ13C excursion identified as the Hirnantian isotope carbon excursion (HICE), which has a magnitude of nearly 2.5‰ above baseline values, is present in an interval from the upper Queenston Formation to the lower to middle part of the Manitoulin Formation in most of Bruce Peninsula and in the area south of Georgian Bay, whereas on Manitoulin Island the HICE appears to be absent. This indicates that a significant part of the Manitoulin Formation is older on the Bruce Peninsula and in its adjacent region than on Manitoulin Island. The chemostratigraphically based conclusions are consistent with biostratigraphic data from conodonts and brachiopods. The Hirnantian δ13C curve from Anticosti Island, Quebec is closely similar to those of southern Ontario. Traditionally, the Ordovician–Silurian boundary has been placed at the base of the Manitoulin Formation, but the new results suggest that it is more likely to be at, or near, the base of the overlying Cabot Head Formation. These new results have major implications for the interpretation of the geologic history and marine depositional patterns of the latest Ordovician of a large part of the North American Midcontinent.