Recently developed methods which allow one to read RNA sequences directly from polyacrylamide gels do not always provide unequivocal results. A combination of primary and secondary in vitro 5′-labeling, as presented here, is methodically and in its results equivalent to fingerprinting and sequencing techniques developed for in vivo labeled RNA. 5 S RNA was used to demonstrate the applicability and reliability of this combination of postlabeling procedures: 5 μg RNA was partially digested, and the resulting overlapping fragments were 5′- 32P-labeled with T4 phage-induced polynucleotide kinase in vitro. After two-dimensional polyacrylamide gel electrophoresis and carrier-free electrophoretic elution, the labeled long fragments, obtained in the 10-ng range, were completely degraded with RNase T 1 and RNase A, respectively. These digests were again 32P-phosphorylated with T4 kinase and lead to fingerprints which allowed the deduction of the nucleotide sequences of the corresponding long fragments.