Type III secretion systems that deliver bacterial proteins into eukaryotic cells are the basis for both symbiotic and pathogenic relationships between many Gram-negative bacteria and their hosts. Exploration of the structure, function and assembly of this secretion system has greatly enhanced our knowledge of bacterial ecology in the context of infectious disease and has spawned new avenues in anti-infective research with a view towards inhibiting virulence functions. We outline advances in understanding type III secretion system function with specific focus on how assembly is hierarchically coordinated at the level of expression and how the type III secretion system mediates transitions in substrate specificity.