Yersinia type III secretion machines transport substrate proteins into the extracellular medium or into the cytoplasm of host cells. Translational hybrids, involving genes that encode substrates as well as reporter proteins that otherwise cannot travel the type III pathway, identified signals that promote transport of effector Yops into host cells. Signals for the secretion of substrates into high calcium media were hitherto unknown. By exploiting attributes of translational hybrids between yopR, whose product is secreted, and genes that encode impassable proteins that jam the secretion machine, we isolated yopR mutations that abolish substrate recognition. Similar to effector Yops, an N-terminal or 5′ signal in codons 1–11 is required to initiate YopR into the type III pathway. YopR secretion cannot be completed and translational hybrids cannot impose a block without a second signal, positioned at codons 131–149. Silent mutations in the second signal abrogate function and the phenotype of other mutations can be suppressed by secondary mutations predicted to restore base complementary in a 3′ stem-loop structure of the yopR mRNA.