Total RNA was prepared from the free polysomes of bovine adrenal cortex and fractionated by high-performance gel filtration chromatography using a TSK-GEL G4000SW or G5000PW column. The former column gave better separation of 28 and 18 S rRNA than did the latter column when a suitable elution condition was selected. The separation of mRNAs by gel filtration chromatography was examined by in vitro translation of the fractionated RNA samples, and the fractionation of mRNAs roughly according to their sizes was confirmed. Significant enrichment of the mRNA of cytochrome P-450(SCC), whose in vitro-synthesized peptide had a molecular weight of 55,000, was achieved.