YscU, a component of the Yersinia type III secretion machine, promotes auto-cleavage at asparagine 263 (N263). Mutants with an alanine substitution at yscU codon 263 displayed secretion defects for some substrates (LcrV, YopB and YopD); however, transport of effector proteins into host cells (YopE, YopH, YopM) continued to occur. Two yscU mutations were isolated that, unlike N263A, completely abolished type III secretion; YscUG127D promoted auto-cleavage at N263, whereas YscUG270N did not. When fused to glutathione S-transferase (Gst), the YscU C-terminal cytoplasmic domain promoted auto-cleavage and Gst-YscUC also exerted a dominant-negative phenotype by blocking type III secretion. Gst–YscUC/N263A caused a similar blockade and Gst–YscUC/G270N reduced secretion. Gst–YscUC and Gst–YscUC/N263A bound YscL, the regulator of the ATPase YscN, whereas Gst–YscUC/G270N did not. When isolated from Yersinia, Gst–YscUC and Gst–YscUC/N263A associated with YscK–YscL–YscQ; however, Gst–YscUC/G270N interacted predominantly with the machine component YscO, but not with YscK–YscL–YscQ. A model is proposed whereby YscU auto-cleavage promotes interaction with YscL and recruitment of ATPase complexes that initiate type III secretion.