Polymerase chain reaction amplification was used to detect catechol 2,3-dioxygenase (CD) gene sequences in the native bacterial populations present in gasoline contaminated groundwater samples. The primers used for the PCR assay were selected from the DNA sequences of the conserved region of the CD gene of xylE, nahH, and pAW313. A 30 bp DNA sequence (ICP313) internal to the structural gene of CD was used as a probe to identify the amplified DNA fragment. The identity of the amplified DNA fragment was confirmed by digesting the DNA fragment with StuI and restriction enzymes which produced DNA fragments of expected sizes. The specificity of the PCR assay was tested using DNA isolated from a variety of bacterial isolates which include Escherichia coli ATCC 25922, Enterobacter faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 29213, Micrococcus roseus ATCC 516, Serratia marcescens ATCC 60, Klebsiella pneumoniae ATCC 13883, Chryseobacterium gleum ATCC 29896, Chromatium vinosum ATCC 17899, Comamonas testosteroni ATCC 11996, Methylosinus trichosporium OB3b, Bacillus subtilis ATCC 21697, Alcaligenes eutrophus ATCC 17698, Arthrobacter globiniformis ATCC 35698, Pseudomonas putida mt-2 ATCC 33015, Acinetobacter calcoaceticus ATCC 31012, and Pseudomonas putida ATCC 17484. Amplification of DNA was not observed in bacterial isolates lacking CD activity. Our primers amplified CD gene sequences in naturally occurring hydrocarbon degrading bacteria isolated from groundwater contaminated with gasoline. The detection limit of the PCR amplification was 10−2 cells of Escherichia coli (pAW313) per ml. A previously characterized hydrocarbon degrader Pseudomonas putida OU83 was used as a positive control. The usefulness of PCR amplification for the detection of CD specific genotypes in the environmental samples was verified using DNA directly extracted from gasoline contaminated groundwater samples as a template. A CD gene specific DNA fragment was amplified from 5 out of 6 gasoline contaminated groundwater samples. The identity and specificity of the PCR amplified DNA generated using the DNA directly extracted from the gasoline contaminated groundwater as template was confirmed by restriction enzyme digestion and Southern hybridization using the 30 bp internal probe (ICP313). Based on the evidence presented in this study, we report for the first time, usefulness of PCR amplification for the detection of CD gene sequences in gasoline contaminated groundwater.