The Escherichia coli phn (psiD) locus consists of a large gene cluster encoding proteins necessary for the use of phosphonates (Pn) as a sole phosphorus source. On the basis of nucleotide (nt) sequence analysis, the phn locus contains a 12.6-kb operon of seventeen genes named, in alphabetical order, phnA to phnQ [Chen et al., J. Biol. Chem. 265 (1990) 4461-4471]. New Pn+ plasmids were made which are suitable for mutational analysis of this gene cluster. These plasmids contain the R6K origin for DNA replication, can be conjugatively transferred, contain the tetAR genes, and therefore provide a way for allele replacement. The construction of these plasmids showed that phnA and phnB have no role in Pn metabolism. Also, these plasmids were employed to introduce nonpolar phnD::lacZ and phnD::uidA fusions into the chromosome, which allowed us to show that phnD probably has a role in transport. In addition, it was shown that phnP is the most distal gene required for Pn use. This was done by testing the effect of phn::uidA insertions in or near the 3' end of phnP on Pn use. Altogether, these results show that all genes required for Pn use are in the 10.9-kb, fourteen-gene, phnCDEFGHIJKLMNOP locus.