Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated Mr of 143,808. The deduced amino acid sequence of the E. coli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.