Five (14)C-radiolabeled azo dyes and sulfanilic acid were synthesized and used to examine the relationship between dye substitution patterns and biodegradability (mineralization to CO2) by a white-rot fungus and an actinomycete. 4-Amino-U-(14)Cbenzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-U-(14)Cbenzenesulfonic acid were used as representative compounds having sulfo groups or both sulfo and azo groups. Such compounds are not known to be present in the biosphere as natural products. The introduction of lignin-like fragments into the molecules of 4-amino-U-(14)Cbenzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-U-(14)Cbenzenesulfonic acid by coupling reactions with guaiacol (2-methoxyphenol) resulted in the formation of the dyes 4-(3-methoxy-4-hydroxyphenylazo)-U-(14)Cbenzenesulfonic acid and 4-(2-sulfo-3'-methoxy-4'-hydroxy-azobenzene-4-azo)-U-(14)C benzenesulfonic acid, respectively. The synthesis of acid azo dyes 4-(2-hydroxy-1-naphthylazo)-U-(14)C benzenesulfonic acid and 4-(4-hydroxy-1-naphthylazo)-U-(14)Cbenzenesulfonic acid also allowed the abilities of these microorganisms to mineralize these commercially important compounds to be evaluated. Phanerochaete chrysosporium mineralized all of the sulfonated azo dyes, and the substitution pattern did not significantly influence the susceptibility of the dyes to degradation. In contrast, Streptomyces chromofuscus was unable to mineralize aromatics with sulfo groups and both sulfo and azo groups. However, it mediated the mineralization of modified dyes containing lignin-like substitution patterns. This work showed that lignocelluloytic fungi and bacteria can be used for the biodegradation of anionic azo dyes, which thus far have been considered among the xenobiotic compounds most resistant to biodegradation. Very specific structural changes in the azo dye molecules enhanced their biodegradability