A method that greatly enhances the detection of tRNA by oligodeoxyribonucleotide probe hybridization has been developed. Because highly structured tRNA regions often preclude heteroduplex formation, we have tested the ability of cold oligodeoxyribonucleotides called unfolders to disrupt the tRNA secondary/tertiary structures and promote hybridization of a second labeled oligonucleotide complementary to the anticodon loop. Here we show that an excess of unfolders in the pre/hybridization reaction can enhance a barely detectable hybridization signal by more than 200-fold without affecting probe specificity. This sensitive assay makes it possible to easily study and monitor changes in tRNA isoacceptor expression.