A 1427-bp DNA fragment containing the kanamycin resistance gene, aphA-3, of plasmid pIP1433 from Campylobacter coli was inserted into a shuttle vector. Full expression of aphA-3 was obtained in Bacillus subtilis and in Escherichia coli. This DNA fragment was sequenced in its entirety and the starting point for aphA-3 transcription in B. subtilis, C. coli and E. coli was determined by S1 nuclease mapping. The sequence of the promoter consists of the hexanucleotides TTGACA and TATAAT, with a spacing of 17 bp. The nucleotide sequence of the aphA-3 gene from C. coli and from the streptococcal plasmid pJH1 are identical whereas they differ by two substitutions and deletion of a codon from that cloned from the staphylococcal plasmid pSH2. These results indicate a recent extension of the resistant gene pool of Gram-positive cocci to Gram-negative bacilli. From an analysis of the DNA sequences surrounding the promoter region, we concluded that the DNA fragment containing the aphA-3 gene in plasmid pJH1 has evolved by deletions from a sequence similar to that found in plasmid pIP1433.