The Willamette River, one of only 14 American Heritage Rivers, flows through the most densely populated and agriculturally productive region of Oregon. Previous biological monitoring of Willamette River fish detected elevated frequencies of skeletal deformities in fish from certain areas of the lower (NP [NP], rivermile [RM] 26-55) and middle (near Wheatland Ferry [WF], RM 72-74) Willamette River, relative to those in the upper Willamette (i.e. near Corvallis [CV], RM 125-138). The objective of this study was to determine the likely cause of skeletal deformities in populations of Willamette River fish. Characterization of deformity loads in Willamette River fish collected in 2002 and 2003 demonstrated that deformity loads remained 2-3 times greater at the NPPool (NP) and WF locations than those observed 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 (≈1-5 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. Chlorpyrifos (averaged less than 1.5 ng/L) was the only organophosphate pesticide detected as bioavailable in water. 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 induced skeletal deformities in cyprinid fish. However, metacercariae of a digenean trematode were directly associated with a large portion of the lesions detected in fish collected from the Willamette River and the lesions were reproduced in fathead minnows exposed to cercariae extracted from field collected snails. As a whole, there was very little evidence to suggest that chemical contaminants were responsible for the greater deformity loads observed at NP and WF. Instead, the weight of evidence suggests that parasitic infection was the primary cause of skeletal deformities observed in Willamette River fish.