Disposable solid-phase microextraction fibers (SPMEs) were used to measure the availability of 2,4,6-trinitrotoluene (TNT) and its two primary transformation products, 2-amino-4,6-dinitrotoluene (2ADNT) and 4-amino-2,6-dinitrotoluene (4ADNT). The SPMEs (85-μm polyacrylate) and sediment-dwelling oligochaetes (Tubifex tubifex) were exposed to TNT-spiked sediment, to TNT-spiked sediment amended with activated carbon, and to TNT-, 2ADNT-, and 4ADNT-spiked water. Sediment concentration was a poor predictor of bioavailability in unamended and carbon-amended sediments (r2 = 0.14–0.73) The activated carbon amendment reduced the bioavailability of compounds in carbon-amended sediment, causing the relationships between Tubifex concentrations and sediment concentrations to differ significantly between unamended and carbon-amended sediment for all compounds. In contrast, SPME TNT concentrations predicted Tubifex TNT concentrations (r2 = 0.54–0.79), and regression models did not differ significantly among the three TNT-spiked matrices. The SPME 2ADNT and 4ADNT concentrations also were predictive of Tubifex 2ADNT and 4ADNT concentrations (r2 = 0.44–0.90). Relationships between Tubifex concentrations and SPME concentrations were the same between unamended and carbon-amended TNT-spiked sediments for 2ADNT and 4ADNT; however, the relationship in sediment (pooled data) differed from the relationship found in 2ADNT- and 4-ADNT-spiked water. The SPMEs provided carbon amendment-independent measures of ADNT availability in sediment and matrix-independent measures of TNT availability among the three matrices. The SPMEs show promise for predicting bioavailable organic compounds in sediment and water.