Many Gram-negative bacteria use a type I secretion system to translocate proteins, including pore-forming toxins, proteases, lipases and S-layer proteins, across both the inner and outer membranes into the extracellular surroundings. The Escherichia coli alpha-hemolysin (HlyA) secretion system is the prototypical and best characterized type I secretion system. The structure and function of the components of the HlyA secretion apparatus, HlyB, HlyD and TolC, have been studied in great detail. The functional characteristics of this secretion system enable it to be used in a variety of different applications, including the presentation of heterologous antigens in live-attenuated bacterial vaccines. Such vaccines can be an effective delivery system for heterologous antigens, and the use of a type I secretion system allows the antigens to be actively exported from the cytoplasm of the bacterial carrier rather than only becoming accessible to the host immune system after bacterial disintegration.