Urinary excretion of nitrophenol metabolites is an important index of human exposure to organophosphate pesticides. In particular, p-nitrophenol, a major urinary metabolite of parathion, can be used as a biomarker of human exposure. Immunoassay methods have been recently described for detection of p-nitrophenol. In the present paper, the effects of a urine matrix on the detection of p-nitrophenol are reported using both fluorescence and absorbance-based ELISAs. A competition format using the enzyme-amplified catalysis of 4-nitrophenyl phosphate or 4-methylumbelliferyl phosphate was used to report the analyte concentration. The presence of urine during the antibody-analyte interaction inhibited product formation in the final step of the assay and shifted the inhibition curves to the right, increasing the apparent I50 values for p-nitrophenol. The various urine samples collected from volunteers, not occupationally exposed to parathion, varied in their ability to inhibit color information and increase I50 values.