A 2,4-dichlorophenoxy acetic acid (2,4-D)-degrading bacterium, strain 58-1, was newly isolated from soil samples collected in the Fukuoka Prefecture, Japan, and grown on an enrichment culture medium containing 2,4-D as the sole carbon source. Phylogenic analysis identified strain 58-1 as Sphingomonas agrestis. In 2,4-D degraders, classes I, II, and III inherit the tfdA, cadA, and tfdAalpha genes, respectively, and the results from degenerate-PCR indicated that this strain belongs to the class II degraders. A clone that includes the cadA gene homolog of S. agrestis 58-1 was screened from a library by using the PCR amplified fragment as a DNA probe. The cloned fragment was sequenced and found to consist of 5043 nucleotides and include 3 open reading frames (orfs). The orf1, orf2, and orf3 genes encode polypeptides consisting of 412, 448, and 177 amino acids, respectively. The Orf2 product shares a high degree of sequence similarity (92%) with the large subunit of 2,4-D oxygenase from the Bradyrhizobium sp. strain HW13, which belongs to the class III 2,4-D degraders, while the orf3 product shared 63% sequence similarity with the small subunit of 2,4-D oxygenase from the strain HW13. The results of the functional expression analysis using various deletion mutants in Escherichia coli revealed that the expression of both orf2 and orf3 genes, but not orf1, is essential for the conversion of 2,4-D to 2,4-DCP. From these results, we conclude the first isolation of 2,4-D oxygenase genes from a class II 2,4-D degrader.