A method for preparation of single-stranded RNA for electron microscopy determination of molecular weight is reported. The method uses treatment with formaldehyde at elevated temperatures to remove secondary structure and spreading in a protein monolayer from 50% formamide onto a 50% formamide hypophase. Molecular weights were determined for some bacterial and animal viruses, for which conflicting values had been reported earlier. Molecular weights determined by the method, using Escherichia coli large subunit rRNA for a standard (1.1 x 10(6)), are as follows: E. coli small subunit rRNA, 0.53 x 10(6); coliphage f2-RNA, 1.3 x 10(6); Qbeta-RNA, 1.55 x 10(6); and Newcastle disease virus RNA, 5.78 x 10(6).