Selected Foraminifera and calcareous nannofossils from the Shubuta Clay, Red Bluff Formation, and Bumpnose Limestone were examined from eight localities and one drilled core in Mississippi and southwestern Alabama, in order to define the biostratigraphic zonation associated with the Eocene-Oligocene boundary. In the northern Gulf Coastal Plain, the Eocene-Oligocene boundary has been placed by previous workers at the disconformable contact of the Shubuta Member of the Yazoo Formation (Jackson Group, uppermost Eocene) and the Red Bluff Formation (Vicksburg Group, lowermost Oligocene), depending upon whether planktonic Foraminifera or nannofossils are used for biostratigraphic zonation. Within the study area, which includes the area between the Mississippi and Tombigbee rivers, the traditional nannofossil Eocene-Oligocene boundary was found to occur at the base of the Shubuta Member of the Yazoo Formation, and the foraminiferal boundary was found to occur at the top of the Shubuta Member. These findings are in agreement with data from Deep Sea Drilling Project Sites, but there is a greater vertical offset between the boundaries in the study area, due to higher sediment accumulation rates in these near-shore marine sediments. It has been suggested by previous studies that perhaps the best solution to the problem of differing Eocene-Oligocene boundaries, dependent upon the microfossil group used to define the boundary, would be to use the "golden spike" principle to define the boundary at the Jackson-Vicksburg contact in the Gulf Coastal Plain, in a section with continuous sedimentation across the boundary. In this study, locality MGS 34 in Wayne County, Mississippi, is suggested as a possible neostratotype.