In crude-oil-contaminant plumes the dissolved organic carbon (DOC) is mainly hydrocarbon degradation intermediates only partly quantified by the diesel range total petroleum hydrocarbon (TPHd) method. To understand potential biological effects of degradation intermediates we tested three fractions of DOC: (1) solid phase extract (HLB); (2) dichloromethane (DCM-total) extract used in TPHd; and (3) DCM extract with hydrocarbons isolated by silica gel cleanup (DCM-SGC). Bioactivity of extracts from five wells spanning a range of DOC was tested using an in vitro multiplex reporter system that evaluates modulation of activity of 46 transcription factors; extracts were evaluated at concentrations equivalent to the well water samples. The aryl hydrocarbon (AhR) and pregnane X (PXR) transcription factors showed the greatest upregulation; with HLB exceeding DCM-total, and no upregulation in the hydrocarbon fraction (DCM-SGC). The HLB extracts were further studied with HepG2 CALUX in vitro assays at nine concentrations ranging from 40 to 0.01 times the well water concentrations. Reponses decreased with distance from the source but were still present at two wells without detectable hydrocarbons. Thus, our in vitro assay results indicate that risks associated with degradation intermediates of hydrocarbons in groundwater will be under estimated when protocols that remove these chemicals are employed.