ABSTRACT In rRNA biogenesis, nuclear myosin 1 (NM1) and actin synergize to activate rRNA gene transcription. Evidence that actin is in preribosomal subunits and NM1 may control rRNA biogenesis post‐transcriptionally prompted us to investigate whether NM1 associates with and accompanies rRNA to nuclear pores (NPC). Ultracentrifugation on HeLa nucleolar extracts showed RNA‐dependent NM1 coelution with preribosomal subunits. In RNA immunoprecipitations (RIPs), NM1 coprecipitated with pre‐rRNAs and 18S, 5.8S, and 28S rRNAs, but failed to precipitate 5S rRNA and 7SL RNA. In isolated nuclei and living HeLa cells, NM1 or actin inhibition and selective alterations in actin polymerization impaired 36S pre‐rRNA processing. Immunoelectron microscopy (IEM) on sections of manually isolated Xenopus oocyte nuclei showed NM1 localization at the NPC basket. Field emission scanning IEM on isolated nuclear envelopes and intranuclear content confirmed basket localization and showed that NM1 decorates actin‐rich pore‐linked filaments. Finally, RIP and successive RIPs (reRIPs) on cross‐linked HeLa cells demonstrated that NM1, CRM1, and Nup153 precipitate same 18S and 28S rRNAs but not 5S rRNA. We conclude that NM1 facilitates maturation and accompanies export‐competent preribosomal subunits to the NPC, thus modulating export.—Obrdlik, A., Louvet, E., Kukalev, A., Naschekin, D., Kiseleva, E., Fahrenkrog, B., Percipalle, P. Nuclear myosin 1 is in complex with mature rRNA transcripts and associates with the nuclear pore basket. FASEB J . 24, 146–157 (2010). www.fasebj.org