A method for efficient nonradioactive labeling of DNA with biotin using random primer extension has been developed. Under the conditions described, a significant amount of DNA synthesis occurs during incorporation of the nonradioactive label, resulting in amplification of the original template DNA. The effect of primer size, substrate concentration, enzyme concentration, and ratio of biotinylated nucleotide to normal nucleotide on the amount of DNA synthesis was determined. Amplifications of 10- to > 300-fold were attained, depending on the starting template concentration. Template may be varied from 1 to 500 ng per reaction. The size of the resulting biotinylated probes is 100-1000 nucleotides with a significant proportion in the 100-300 nucleotide range. The biotinylated probes were used to detect single-copy genes on Southern blot hybridizations and to identify specific loci in metaphase chromosome spreads by in situ hybridization followed by fluorescent detection with streptavidin-fluorescein isothiocyanate. Random primer amplification and labeling provides a convenient method for preparation of biotinylated probes from small amounts of template DNA.