Members of the LacI family of transcriptional repressors respond to the presence of small effector molecules. The binding of the ligands affect the proteins ability to repress transcription by stabilizing a conformation that, in most cases, is unfavorable for high-affinity DNA binding. The CytR anti-activator diverges from the other family members by relying on the cooperative DNA binding with the global regulator CRP. The inducers of CytR do not affect CytR-DNA binding per se, but alleviate repression by interrupting protein-protein interactions between the two regulators. Here, we have studied of the CytR-inducer interaction by exploring a discrepancy in the inducer response observed for the homologous CytR regulators of Escherichia coli and Salmonella typhimurium. CytR of S. typhimurium (CytR(St)) appears to respond to the presence of both uridine and cytidine nucleosides, whereas E. coli CytR (CytR(Ec)) responds to cytidine only. We have used a combination of genetic and structural modeling studies to provide detailed information regarding the nature of this discrepancy. By analysis of hybrid CytR proteins followed by site-directed mutagenesis, we have successfully transferred the specificity determinants for uridine from CytR(St) to CytR(Ec), revealing that serine substitutions of only two residues (G131 and A152) in CytR(Ec) is required to make CytR(Ec) sensitive to uridine. In addition, by employing a genetic screen for induction of defective mutants, we have identified four amino acid residues in CytR(St) that appear to be important for the response to uridine. The implications of these findings for the understanding of the ligand binding and induction of CytR are discussed in the context of the structural knowledge of CytR and homologous protein-ligand complexes.