ortho-Nitrophenyl-ò-galactoside (ONPG) is a colorimetric and spectrophotometric substrate for detection of ò-galactosidase activity. This compound is normally colorless. However, if ò-galactosidase is present, it hydrolyzes the ONPG molecule into galactose and ortho-nitrophenol. The latter compound has a yellow color that can be used to check for enzyme activity by means of a colorimetric assay (at 420 nm wavelength). ò-Galactosidase is required for lactose utilization, so the intensity of the color produced can be used as a measure of the enzymatic rate.
Though ONPG mimics lactose and is hydrolyzed by ò-galactosidase, it is unable to act as an inducer for the lac operon. Without another lactose analog that can act as an inducer, such as isopropyl ò-<small>D</small>-1-thiogalactopyranoside (IPTG), ò-galactosidase will not be transcribed and ONPG will not be hydrolyzed.