This report describes a novel technique for the preparation of homogeneously labeled DNA probes. The procedure uses a heteroduplex intermediate, which is directly prepared from a double-stranded plasmid, and therefore requires neither sub-cloning of the fragment of interest into a specialized vector nor the use of a synthetic primer. The technical advantages of the procedure are demonstrated by preparation of a probe able to determine the precise location and efficiency of utilization of the polyadenylation site used during transcription of the rat preproinsulin II gene in a transfected avian cell line.