About 1500 hybrid broad-host-range plasmids from a genomic library of Pseudomonas saccharophila were individually transferred by conjugation from Escherichia coli to Alcaligenes eutrophus. Direct selection for pentose-utilizing transconjugants yielded three clones capable of growth on xylose. Growth of P. saccharophila as well as the transconjugants of A. eutrophus on xylose was relatively slow, exhibiting doubling times of about 9.5 h. Plasmid pGN3 harbored by one transconjugant contained a 28-kb DNA insert, 16.4 kb of which comprised the minimal information required for xylose utilization by A. eutrophus. At least the xyl genes encoding xylose isomerase and xylulokinase were located within this region, as indicated by their induction during growth of A. eutrophus (pGN3) on xylose. Southern hybridizations with a heterologous gene probe confirmed the presence of these xyl genes. In both P. saccharophila and A. eutrophus (pGN3), low activities of several enzymes operating in the pentose phosphate and Entner-Doudoroff pathways might limit the rate of xylose catabolism.