The potential use of a class-specific enzyme-linked immunosorbent assay (ELISA) in studying the occurrence and fate of tetracyclines in the environment was evaluated. Several manure samples collected from hog lagoons and cattle feedlots were screened for the presence of tetracycline residues using ELISA. The levels varied from less than the detection limit (0.5 parts per billion) to 200 parts per million. The degradation of tetracyclines in soil-applied manure was followed using ELISA to measure the decline in tetracycline concentrations. Low levels of tetracyclines remained detectable in soil for up to 28 days. The ELISA procedure also proved useful in determining the leaching potential of tetracyclines in undisturbed soil columns and in the analysis of total tetracyclines in manure, soil, and water. Based on the cross-reactivity of the antibodies employed, this ELISA method can be an important screening tool for the presence of other tetracycline compounds, such as chlortetracycline and oxytetracycline. The ELISA method also detects the epimers of tetracyclines and the corresponding dehydration by-products, anhydrotetracyclines. Analysis of selected manure extracts by liquid chromatography with mass spectrometry (LC-MS) showed lower concentrations of total tetracyclines compared to the values obtained by ELISA, indicating the presence of other structurally related compounds or transformation products of tetracyclines being detected by ELISA in the samples. Because analysis of manure and soil samples by LC-MS requires extensive clean-up procedures, ELISA provides an alternative method for conducting environmental fate and transport studies of antibiotics.