The nucleotide sequence was established for the aphVIII aminoglycoside phosphotransferase gene of an oxytetracycline-producing Streptomyces rimosus strain. The gene is 804 bp in size and possibly codes for APHVIII of 267 residues. Heterologous expression of aphVIII was studied in Escherichia coli and Chlamydomonas reinhardtii. The deduced APHVIII sequence was compared with known sequences of aminoglycoside phosphotransferases of aminoglycoside-producing actinomycete strains and of eukaryotic protein kinases. A local homology of 38 residues was found between APHVIII and actinomycete serine-threonine protein kinases in the conserved region possibly involved in ATP binding. APHVIII differed from aminoglycoside 3'-phosphotransferases of aminoglycoside-producing actinomycete strains and of clinical isolates, and can be classed to a separate group.