A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N′ (N-d-biotinyl-3-aminopropyl)-N′-methyl-1,3-propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100–400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 × 10⁻¹⁸ mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with ³²P-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.