5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside is most commonly known asX-Gal. X-gal is a histochemical substrate for β-galactosidase which cleaves X-Gal to yield an insoluble blue precipitate.

X-Gal is a mainstay in molecular biology cloning applications in which it is used to detect the activity of β-galactosidase. X-Gal is used to detect the insertion of foreign DNA into the lacZ region of a plasmid DNA. Insertion of DNA into the lacZ region results in the loss of β-galactosidase activity. Bacteria cells that retain active β-galactosidase will result in characteristic blue colonies. Successful disruption of thelacZαgene disrupts the α-complementation of the β-galactosidase gene and the precipitate does not form, resulting in white colonies.

Many other applications also use X-Gal as a substrate to detect β-galactosidase activity. These include β-galactosidase -antibody linked immunoassays and immunohistochemistry, coliphage detection based on β-galactosidase induction and the detection of micrometastasis formation during tumor progession.