The periplasm is a region in the cell wall of Gram-negative bacteria. It is located between the outer membrane and the inner, or cytoplasmic, membrane. Once considered to be empty space, the periplasm is now recognized as a specialized region of great importance.

The existence of a region between the membranes of Gram-negative bacteria became evident when electron microscopic technology developed to the point where samples could be chemically preserved, mounted in a resin, and sliced very thinly. The so-called thin sections allowed electrons to pass through the sample when positioned in the electron microscope. Areas containing more material provided more contrast and so appeared darker in the electron image. The region between the outer and inner membranes presented a white appearance. For a time, this was interpreted as being indicative of a void. From this visual appearance came the notion that the space was functionless. Indeed, the region was first described as the periplasmic space.

Techniques were developed that allowed the outer membrane to be made extremely permeable or to be removed altogether while preserving the integrity of the underlying membrane and another stress-bearing structure called the peptidoglycan. This allowed the contents of the periplasmic space to be extracted and examined.

The periplasm, as it is now called, was shown to be a true cell compartment. It is not an empty space, but rather is filled with a periplasmic fluid that has a gel-like consistency. The periplasm contains a number of proteins that perform various functions. Some proteins bind molecules such as sugars, amino acids, vitamins, and ions. Via association with other cytoplasmic membrane-bound proteins these proteins can release the bound compounds, which then can be transported into the cytoplasm of the bacterium. The proteins, known as chaperons, are then free to diffuse around in the periplasm and bind another incoming molecule. Other proteins degrade large molecules such as nucleic acid and large proteins to a size that is more easily transportable. These periplasmic proteins include proteases, nucleases, and phosphatases. Additional periplasmic proteins, including beta lactamase, protect the bacterium by degrading incoming antibiotics before they can penetrate to the cytoplasm and their site of lethal action.

The periplasm thus represents a buffer between the external environment and the inside of the bacterium. Gram-positive bacteria, which do not have a periplasm, excrete degradative enzymes that act beyond the cell to digest compounds into forms that can be taken up by the cell.