In the primer extension assay, the transcription start site for a gene is determined experimentally by identifying the 5' end of the encoded messenger RNA (mRNA). The protocol begins with a primer, usually a synthetic oligonucleotide of about 20 residues, that is complementary to an mRNA sequence ∼50-150 nucleotides downstream of the anticipated 5' end. The primer is 5'-end-labeled using [γ-(32)P]ATP and T4 polynucleotide kinase and is annealed to the specific mRNA molecules within an RNA sample. Reverse transcriptase (RT), deoxyribonucleoside triphosphates, and appropriate buffer components are added to the primer-mRNA hybrids to catalyze elongation of the primer to the 5' end of the mRNA. The resulting radiolabeled complementary DNA (cDNA) products are analyzed by denaturing polyacrylamide gel electrophoresis, followed by autoradiography. The sizes of the bands detected on the gel, as compared to an adjacent sequencing ladder or molecular weight standards, provide a measure of the distance from the 5' end of the synthetic oligonucleotide to the beginning of the mRNA transcripts. In theory, the 3' end of the cDNA will coincide with the 5' end of the mRNA. Thus, the size of the radiolabeled cDNAs should represent the distance from the labeled 5' end of the primer to the 5' end of the mRNA (i.e., the 3' end of the cDNA). If the labeled cDNA products are within the resolution range of the gel, the transcription start site can be determined with an accuracy of plus or minus one nucleotide.