The authors established a simple, rapid and effective procedures for sequencing the targeted a-gene SupF TRNA of shuttle vector pSP189, which is very important and useful in molecular mechanism research of mutagens. In this method, double-stranded plasmid DNA as template, prepared single-stranded DNA with alkali denaturation, applied r-32P-ATP for labelling 5'-end of primer, were used. A satisfactory result was achieved.