Because of their low abundance and short length, telomeric single-stranded extensions have not traditionally been assessed by Southern blot analysis. Instead, most methods have relied on hybridizing radioactively labeled oligonucleotide probes to electrophoresed DNA within agarose gels. Here we describe a rapid and nonradioactive Southern blot-derived method to transfer and detect telomeric single-stranded G-rich overhangs (G-tails) under nondenaturing (native) conditions, using Saccharomyces cerevisiae DNA. Restriction enzyme-digested chromosomal DNA is separated by agarose gel electrophoresis, transferred onto a charged membrane by electroblotting under nondenaturing conditions, and probed with a digoxigenin (DIG)-labeled oligonucleotide. Compared with the prolonged film exposure required to detect radioactive probes, detection of short single-strand G-tails with this method takes mere minutes. Furthermore, following detection of the single-stranded G-tails, the DNA on the membrane can be denatured and reprobed using conventional hybridization and detection methods.