For sulfonamides, the formation of non-extractable residues has been identified by laboratory testing as the most relevant concentration determining process in manured soil. Therefore, the present study has been focused on the chemical and biological characterization of non-extractable residues of (14)C-labeled sulfadiazine or sulfamethoxazole. In laboratory batch experiments, the test substances were spiked via standard solution or test slurry to microbially active soil samples. After incubation periods of up to 102d, a sequential extraction technique was applied. Despite the exhaustive extraction procedure, sulfadiazine residues mainly remained non-extractable, indicating the high affinity to the soil matrix. The remobilization of non-extractable (14)C-sulfadiazine residues was monitored in the activated sludge test and the Brassica rapa test. Only small amounts (<3%) were transferred into the extractable fractions and 0.1% was taken up by the plants. In the Lumbricus terrestris test A, the release of non-extractable (14)C-sulfamethoxazole residues by the burrowing activity of the earthworms was investigated. The residues mainly remained non-extractable (96%). The L. terrestris test B was designed to study the immobilization of (14)C-sulfamethoxazole in soil directly after the test slurry application. The mean uptake by earthworms was 1%. Extractable and non-extractable residues amounted to 5% and 93%, respectively. Consequently, the results of all tests confirmed the high affinity of the non-extractable sulfonamide residues to the soil matrix.