The ftsYEX operon in Escherichia coli encodes three proteins, two of which (FtsE and FtsX) are known to be required for cell division. Although FtsE and FtsX have been identified using SDS-PAGE, the FtsY protein has not. We have used in vitro insertion mutagenesis to identify FtsY as a 92 kD polypeptide in maxicell experiments, although predictions from the DNA sequence estimated FtsY to be 54 kD. Results suggest that this disparity could be due to the unusually high percentage of acidic residues within the protein. Complementation tests indicated the presence of a promoter within ftsY required for expression of ftsE and ftsX. The FtsY protein exhibits sequence homology with the SR alpha protein of eukaryotes which is involved in protein secretion. The essential nature of the ftsY gene was also demonstrated.