Activation of bacterial virulence-associated type III secretion systems (T3SSs) requires direct contact between a bacterium and a eukaryotic cell. In Y. pestis, a cytosolic YopN-TyeA complex functions to block type III secretion (T3S) in the presence of extracellular calcium and prior to contact with a eukaryotic cell. The mechanism by which the bacterium senses extracellular calcium and/or cell contact and transmits these signals to the cytosolic YopN-TyeA complex is unknown. I report here that YscF, a small protein that polymerizes to form the external needle of the T3SS, is essential for the cell contact- and calcium-dependent regulation of T3S. Alanine-scanning mutagenesis was used to identify YscF mutants that secrete virulence proteins in the presence and absence of calcium and prior to contact with a eukaryotic cell. Y. pestis YscF mutants with amino acid replacements I13A, D28A and D46A resulted in constitutive (unregulated) secretion that required 2.5, 5 and 7.5 mM Ca2+, respectively, to block secretion. Interestingly, a YscF D28A D46A double mutant that exhibited constitutive T3S that could not be blocked by any amount of Ca2+ was unable to translocate secreted Yops across a eukaryotic membrane. These data indicate that the YscF needle is a multifunctional structure that participates in virulence protein secretion, in translocation of virulence proteins across eukaryotic membranes and in the cell contact- and calcium-dependent regulation of T3S.