The presence of dioxin-like compounds in sediments from harbors and reference sites along the Dutch coast was investigated using the dioxin receptor–chemically activated luciferase gene expression (DR-CALUX) bioassay. The DR-CALUX response varied between 0.2 and 136 ng/kg dry weight expressed in units of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalency quotients (TEQ). The highest CALUX-TEQ values (>50 ng TEQ/kg dry wt) were found in sediments from the center of the Port of Rotterdam and from the North Sea canal near Amsterdam. The DR-CALUX activity of harbor sediments was classified by setting guideline values. None of the 257 harbor sediment samples investigated exceeded the maximum value of 1,000 ng TEQ/kg, while 94% of the samples fail the target value of 2 ng TEQ/kg. Threshold values (25 and 50 ng TEQ/kg) are intended as pass/fail criteria for offshore disposal of dredged material and were exceeded in 12 and 3% of the samples, respectively. DR-CALUX response did not always match with contamination of polychlorinated biphenyls (PCBs) as determined in bulk sediments (Σ7-PCB ranging from <1 to 456 μg/kg dry wt). Concentrations of planar PCBs, polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated-dibenzofuranes (PCDFs), polybrominated biphenyls (PBBs), and polybrominated-diphenylethers (PBDEs) were determined in 20 selected sediment extracts. On average, PCDD/Fs explained 50% of the DR-CALUX activity and planar PCBs 6%. Further research is needed to elucidate the unexplained part of the DR-CALUX response. The DR-CALUX is a highly sensitive and reproducible bioassay that can usefully complement standard PCB analysis, improving the hazard assessment of the disposal of dredged material in the North Sea.