Random genomic DNA fragments from Saccharomyces cerevisiae were tested for their ability to activate transcription of a promoterless aminoglycoside phosphotransferase-encoding gene in Streptomyces. About 10% of the insertions led to kanamycin resistance when selected at low concentration (5 micrograms ml-1). The nucleotide sequences of five insertions that allowed growth at different concentrations of the antibiotic were determined. Three of them contained -10 and -35 consensus sequences for the major class of eubacterial promoters. In two others, a -10 sequence could be identified, but a -35 element was absent at the appropriate distance. All of the five inserts were also transcriptionally active in Escherichia coli and therefore probably belong to the major class of eubacterial promoters. Three of the characterized insertions found to match known yeast sequences did not derive from promoter regions. We conclude that sequences that function as eubacterial promoters occur at random in the yeast genome.