Contraction of muscles is mediated by highly organized arrays of myosin motor proteins. We report here the characterization of a mutation of a UCS gene named steif/unc-45b that is required for the formation of ordered myofibrils in both the skeletal and cardiac muscles of zebrafish. We show that Steif/Unc-45b interacts with the chaperone Hsp90a in vitro. The two genes are co-expressed in the skeletal musculature and knockdown of Hsp90a leads to impaired myofibril formation in the same manner as lack of Steif/Unc-45b activity. Transcripts of both genes are up-regulated in steif mutants suggesting co-regulation of the two genes. Our data indicate a requirement of Steif/unc-45b and Hsp90a for the assembly of the contractile apparatus in the vertebrate skeletal musculature.