Phenazine compounds produced by P. aeruginosa play important roles in pathogenicity as virulence factors as well as signaling molecules. Previously, we have identified genes that altered phzA1 expression in the presence of subinhibitory concentrations of tetracycline. In this study, we found both phzA1 and phzA2 were activated by subinhibitory concentrations of spectinomycin (Spc). Using luxCDABE based reporters we screened transposon libraries and identified 12 genes whose mutation changed the activation of phzA1 and/or phzA2 by Spc, and therefore potentially involved in phenazine regulation. Among these genes, the interruption of lasI, pqsR and pqsE abolished phzA1 and phzA2 expression while the mutation of vqsM stopped phzA2 expression, and therefore Spc lost its effect in all these mutants. The disruption of 3 fimbrial related genes, pilY, pilX, and pilH independently increased the degrees of Spc activation of phzA2 whereas the algC and truA mutations decreased the Spc activation of phzA2. Ppk and pcnB mutants had increased Spc activation of phzA1 expression. Complementation assays with cloned genes carried on a plasmid in the truA mutant restored the Spc activation of phzA2, confirming the involvement of this gene in Spc regulation of phzA2 operon. The phzA1 expression in the presence of Spc was only partially restored by pcnB complementation, suggesting other genes in the pcnB operon may play a role. The information obtained in this study provides a basis for further investigations.