Ring-hydroxylating dioxygenases (RHDs) are of central importance to bacterial recycling of aromatic hydrocarbons, including anthropogenic pollutants. The database of presently characterized RHDs is biased towards those from organisms readily isolated on anthropogenic substrates. To investigate the extent to which RHDs from extant organisms reflect the natural diversity of these enzymes, we developed a polymerase chain reaction (PCR) method for retrieval of RHD gene fragments from environmental samples. Gene libraries from two contaminated and two pristine soil samples were constructed. None of the inferred peptides from clones examined were identical to previously described RHDs; however, all showed significant sequence homology and contained key catalytic residues. On the basis of sequence identity, the environmental clones clustered into six distinct groups, only one of which included known RHDs. One of the new sequence groupings was particularly widespread, being recovered from all soil samples tested. Comparison of inferred peptide sequences of the environmental clones and known RHDs showed the former to have greater sequence variation at sites thought to influence accessibility of the active site than that seen between currently known RHDs. We conclude that presently characterized RHDs do not adequately represent the diversity of function found in in situ forms.