The polarity suppression (Psu) protein of bacteriophage P4 causes suppression of transcriptional polarity inEscherichia coliby overcoming Rho termination factor activity. Two newpsumutants defective in polarity suppression are described. Thepsu5mutation deletes codons 95–98 from about the middle of the gene, and the mutant protein is inactive. Thepsu6mutation changes Phe169 to Val and encodes a temperature-sensitive protein. Constitutive overexpression ofpsu+from a plasmid prevents colony formation, but overexpression of mutant genes (psu5, psu6) does not, suggesting that Psu disturbs essential host function(s). Rho protein synthesis is enhanced several-fold in cells containing wild-type Psu, due to readthrough at therhoattenuator, while the physical stability of Rho is maintained. As a consequence, Psu-producing cells accumulate significantly more Rho than normal cells, reminiscent of termination-defectiverhomutants. The polarity suppression activity induced by Psu is demonstratedin vitroby the efficient readthrough of Rho-dependent terminators λ tR1and TIS2during coupled transcription–translation. Purified Rho protein restores termination at TIS2when added to Psu-containing reactions but NusG does not. The data support the hypothesis that Psu has or elicits an anti-Rho function.