Strains of Pseudomonas aeruginosa causing pulmonary infections in cystic fibrosis patients have an unusual mucoid phenotype because of production of the capsule-like exopolysaccharide, alginate. Transcriptional activation of algD, the first gene of a large alginate biosynthetic gene cluster, is associated with conversion to the alginate-producing (Alg+) phenotype. In this study, we examined the regulation of alginate genes immediately downstream of algD. Mutants of the Alg+ strain FRD1 were constructed by gene replacement with defined Tn501 (8.2kb) insertions in the alginate biosynthetic gene cluster, resulting in an Alg- phenotype. The Alg+ phenotype of these mutants was restored by integration of narrow-host-range plasmids containing DNA fragments from P. aeruginosa that reconstructed a continuous alginate gene cluster. A broad-host-range plasmid containing the entire alginate gene cluster except for the terminal gene, algA, was unable to complement an alG::Tn501 mutant unless algA was transcribed from a second plasmid. This indicated that any Tn501 insertion in the cluster was polar on downstream alginate genes. Northern blot hybridization experiments also showed that a transposon insertion downstream of algD adversely affected algG and algA transcription. These results provided evidence that the alginate biosynthetic gene cluster has an operonic structure and is cotranscribed from the algD promoter.