The Albemarle-Pamlico Estuarine System (APES) is the second largest estuarine system within the mainland of the United States and is estimated to have lost about half of its submerged aquatic vegetation (SAV) over the past several decades. The issue of herbicide runoff and subsequent toxic effects to SAV is important because of the extensive agricultural production that occurs in the APES region. The aim of this study was to conduct a retrospective analysis of herbicide influx to waters of the APES region during the time period of documented SAV declines and to compare the measured concentrations to SAV toxicity thresholds and changes in agricultural land use. Surface water grab samples were collected at 26 sites in the APES region during May through July 2000. The most consistently measured herbicides were alachlor, atrazine, and metolachlor with geometric mean concentrations ranging from 29 to 2463 ng/L for alachlor, 14 to 7171 ng/L for atrazine, and 17 to 5866 ng/L for metolachlor. Concentrations of alachlor, atrazine, and metolachlor measured in water samples from the APES region in 2000 exceeded several of the established benchmarks, standards, or guidelines for protection of aquatic plants. Although this evaluation was of point-in-time herbicide samples (year 2000) and not analyzed for all possible herbicides used at the time, they were taken during the period of SAV declines, reveal the plausibility of exposure risk to SAV, and suggest that herbicide runoff should be studied along with other variables that influence SAV growth and distribution in future studies.