Yeast RNA:pseudouridine synthetase Pus1 catalyzes the formation of pseudouridines in tRNAs. We report here the quaternary structure of purified recombinant Pus1 in solution. At low concentration, in the absence of tRNA, Pus1 oligomerizes while at high concentration it precipitates. This oligomerization/aggregation can be prevented by addition of dodecyl-beta-D-maltoside or of yeast tRNA(Phe). The detergent does not significantly interfere with substrate binding or with activity of Pus1. The stoichiometry of the Pus1/tRNA(Phe) complex is 1/1. We conclude that the detergent covers an hydrophobic region of the RNA binding pocket responsible for Pus1 aggregation.