A genomic library of total DNA of Pseudomonas cepacia AC1100 was constructed on a broad-host-range cosmid vector pCP13 in E. coli AC80. A 25-kb segment was isolated from the library that complemented a Tn5-generated, 2,4,5-T minus mutant, P. cepacia PT88. A simple colorimetric assay was developed to detect the presence of this active enzyme in intact cells and was used to determine the expression of complementing genes. Subcloning experiments showed that a 4-kb BamHI-PstI fragment and a 290-bp PstI-EcoRI fragment, separated by 1.3-kb, were required for complementation. Both fragments were identified to be chromosomal in origin. Hybridization studies using the subcloned fragments revealed that in addition to a Tn5 insertion, mutant PT88 contained an extensive chromosomal deletion accounting for its 2,4,5-T-phenotype. The cloned fragments did not show homology to plasmid DNAs carrying degradative genes for toluene, naphthalene, and 3-chlorobenzoate. (Copyright 1988 Elsevier Science Publishers B.V. (Biomedical Division).)