We have examined the effect of the F plasmid TraY protein on tm gene expression in vivo. Expression was assayed as alkaline phosphatase activity in cells containing a traY Phi(traA'-'phoA)hyb operon under traY promoter control. Amber mutations in traY significantly reduced alkaline phosphatase activity. Since nonsense polarity effects were minimal, if they occurred at all, these data provide the first direct evidence that TraY regulates tra gene expression.