The Willamette River, one of 14 American Heritage Rivers, flows through the most densely populated and agriculturally productive region of Oregon. Previous biological monitoring of the Willamette River detected elevated frequencies of skeletal deformities in fish from certain areas of the lower (Newberg pool [NP], rivermile [RM] 26−55) and middle (Wheatland Ferry [WF], RM 72−74) river, relative to those in the upper river (Corvallis [CV], RM 125−138). The objective of this study was to determine the likely cause of these skeletal deformities. In 2002 and 2003, deformity loads in Willamette River fishes were 2−3 times greater at the NP and WF locations than at the CV location. There were some differences in water quality parameters between the NP and CV sites, but they did not readily explain the difference in deformity loads. Concentrations of bioavailable metals were below detection limits (0.6−1 μg/L). Concentrations of bioavailable polychlorinated biphenyls (PCBs) and chlorinated pesticides were generally below 0.25 ng/L. Concentrations of bioavailable polycyclic aromatic hydrocarbons were generally less than 5 ng/L. Concentrations of most persistent organic pollutants were below detection limits in ovary/oocyte tissue samples and sediments, and those that were detected were not significantly different among sites. Bioassay of Willamette River water extracts provided no evidence that unidentified compounds or the complex mixture of compounds present in the extracts could induce skeletal deformities in cyprinid fish. However, metacercariae of a digenean trematode were directly associated with a large percentage of deformities detected in two Willamette River fishes, and similar deformities were reproduced in laboratory fathead minnows exposed to cercariae extracted from Willamette River snails. Thus, the weight of evidence suggests that parasitic infection, not chemical contaminants, was the primary cause of skeletal deformities observed in Willamette River fish.