Previous studies provided evidence that nonstructural protein muNS of mammalian reoviruses is present in particle assembly intermediates isolated from infected cells. Morgan and Zweerink (Virology 68455-466, 1975) showed that a subset of these intermediates, which can synthesize the viral plus strand RNA transcripts in vitro, comprise core-like particles plus large amounts of muNS. Given the possible role of muNS in particle assembly and/or transcription implied by those findings, we tested whether recombinant muNS can bind to cores in vitro. The muNS protein bound to cores, but not to two particle forms, virions and intermediate subvirion particles, that contain additional outer-capsid proteins. Incubating cores with increasing amounts of muNS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of muNS were bound. Thus, the muNS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the muNS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to muNS. The muNS-core complexes retained the capacity to synthesize the viral plus strand transcripts as well as the capacity to add methylated caps to the 5' ends of the transcripts. In vitro competition assays showed that mixing muNS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins mu1 and sigma3. These findings are consistent with the presence of muNS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, muNS may block or delay outer-capsid assembly and allow continued transcription by these particles.