We have developed a precise and convenient mapping technique for determining transcription start points (tsp) on cloned genomic DNA using T4 DNA polymerase. This method uses single-stranded (ss) M13 DNA and therefore is, unlike S1 and Exo VII nuclease mapping methods, independent of the restriction endonuclease sites present in the insert. Essentially the protocol involves the following steps: hybridizing an mRNA to an ss M13 vector containing an antisense genomic DNA sequence spanning the presumptive tsp (cap site); annealing a DNA primer (M13 sequencing primer) to the M13 DNA at a site on this DNA upstream from the 5' end of the mRNA on the template DNA; extending the DNA primer with T4 DNA polymerase towards the 5' end of the mRNA. Since T4 DNA polymerase will not displace the mRNA: DNA hybrid, synthesis is blocked at the first nucleotide of the mRNA molecule. The length of the extended DNA products can then be determined with single nucleotide resolution on denaturing sequencing gels in parallel with a sequencing ladder. We have used this approach to map the tsp of the mouse skeletal alpha-actin gene. The sensitivity of the method allows precise mapping of transcripts present as 0.02-0.05% of the total RNA. This method is particularly valuable for mapping the tsp of genes which are known to contain a large intron between the first and second exons. It can also be applied to map the 5' border of any given exon of a gene in an M13 vector or in other vectors that give ss DNAs.