The pldA gene of Escherichia coli K12, which is involved in the synthesis of an outer membrane (OM) phospholipase, has been cloned using a cosmid cloning system. For detection of the cloned gene a newly developed, in vivo phospholipase assay was used. Subsequent cloning of the pldA gene was performed into the multicopy plasmid vectors pBR322 and pACYC184. The gene was localised on these hybrid plasmids by the analysis of in vitro-constructed deletion plasmids and mutant plasmids generated by transposon gamma delta-insertions. Analysis of plasmid-encoded proteins in a minicell system showed that the pldA gene product is a polypeptide with apparent molecular weight of 29,000. This apparent molecular weight changes from 29,000 to 26,000 when the denaturing temperature is changed from 95 degrees C to 37 degrees C. These data are in agreement with those on purified OM phospholipase (Nishijima et al. 1977), and therefore strongly suggest that pldA is the structural gene for this phospholipase. From the minicell experiments the direction of transcription of pldA could be established relative to the metE gene, which is also cloned on the same hybrid plasmids. Strains carrying the pldA gene on these high copy vectors do not appear to be affected by the product with respect to cell growth in any way. However they do harbour increased amounts of 29 K protein in cell envelope fractions, indicating that gene expression and product translocation to the OM are proportional to the increased gene copy number. We therefore conclude that phospholipase enzymatic activity is strictly regulated at the protein level.