The Maxam-Gilbert method of nucleotide sequence determination is based on preferential, base-specific methylation followed by chemical cleavage to generate a nested set of end- labeled derivatives.

In 1976-1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases. Although Maxam and Gilbert published their chemical sequencing method two years after the ground-breaking paper of Sanger and Coulson on plus-minus sequencing, Maxam-Gilbert sequencing rapidly became more popular, since purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA. However, with the improvement of the chain-termination method (see below), Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity prohibiting its use in standard molecular biology kits, extensive use of hazardous chemicals, and difficulties with scale-up.

The method requires radioactive labelling at one end and purification of the DNA fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first 'cut' site in each molecule. The fragments in the four reactions are arranged side by side in gel electrophoresis for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabelled DNA fragment, from which the sequence may be inferred.

Also sometimes known as 'chemical sequencing', this method originated in the study of DNA-protein interactions (footprinting), nucleic acid structure and epigenetic modifications to DNA, and within these it still has important applications.