Two different monoclonal antibodies (MAb) were raised against 2,4-dichlorophenol hydroxylase (DCP-hydroxylase) of Ralstonia eutropha JMP134 (pJP4), the second enzyme in the 2,4-D-degradative pathway of this bacterium. The utility of these antibodies in detecting and characterizing 2,4-D-degrading soil bacteria was investigated. One MAb (F6) reacted with DCP-hydroxylase from 27 out of 36 strains tested, while the other (MAb C3) reacted with only 17 isolates. When used with the colony blot technique, MAb F6 was useful for detecting cross-reacting strains on plates of pure cultures or of mixtures containing nondegraders even when 2,4-D degraders were outnumbered 60 to 1. 2,4-D-degrading strains could also be detected from plates spread with enrichment cultures but not from primary isolation plates spread from soil dilutions, presumably because the ratio of degraders to nondegraders was too low. Colonies of some strains that were very distantly related genetically, but produced functionally similar DCP-hydroxlase enzymes, were detected by MAb F6. This result suggests that MAbs could be useful for detecting functionally similar proteins expressed from tfdB analogs, even in the absence of detectable DNA homology between the genes encoding them.