We previously identified a Pseudomonas sp. strain, ADP, which rapidly metabolized atrazine in liquid culture, agar plates, and soils (R. T. Mandelbaum, D. L. Allan, L. P. Wackett, Appl. Environ. Microbiol. 61:1451-1457, 1995). In this study, we report the cloning and partial characterization of a gene region from Pseudomonas sp. strain ADP that encodes atrazine degradation activity. A 22-kb EcoRI genomic DNA fragment, designated pMD1, was shown to encode atrazine dechlorination activity in Escherichia coli DH5 alpha. Atrazine degradation was demonstrated by a zone-clearing assay on agar medium containing crystalline atrazine and by chromatographic methods. A gene conferring the atrazine-clearing phenotype was subsequently subcloned as a 1.9-kb AvaI fragment in pACYC184, designated pMD4, and was expressed in E. coli. This result and random Tn5 mutagenesis established that the 1.9-kb AvaI fragment was essential for atrazine dechlorination. High-pressure liquid and thin-layer chromatographic analyses were used to rigorously establish that E. coli containing pMD4 degraded atrazine and accumulated hydroxyatrazine. Hydroxyatrazine was detected only transiently in E. coli containing pMD1. This is consistent with the idea that hydroxyatrazine is the first metabolite in atrazine degradation by Pseudomonas sp. strain ADP. A 0.6-kb ApaI-PstI fragment from pMD4, containing the putative atrazine chlorohydrolase gene, hybridized to DNA from atrazine-degrading bacteria isolated in Switzerland and Louisiana.(ABSTRACT TRUNCATED AT 250 WORDS)