A method which allows discrete nucleic acid sequences to be detected with differently colored hybridization signals on the same blot involving only a single hybridization step is described. Nucleic acid probes labeled with digoxigenin, fluorescein, or biotin are hybridized simultaneously to immobilized target nucleic acids. Differential colorimetric detection is carried out in consecutive alkaline phosphatase-based immunoassays with one of three 3-hydroxy-2-naphthoic acid anilide phosphate/diazonium salt combinations as substrate. Each label is visualized by a different color precipitate (green, red, and blue) directly on the membrane. We demonstrate the use of this method in multicolor plasmid mapping, detection of different genomic sequences on a single Southern blot, discrimination of transcription levels in a Northern blot, and colony screening. Advantages and limitations of the method, as well as further applications, are discussed.