Recombinant plasmids carrying either the wildtype kanamycin nucleotidyltransferase gene encoded originally by the mesophilic plasmid pUB110 or the gene encoding the thermostable TK101 mutant were constructed and introduced into Bacillus stearothermophilus by a protoplast transformation procedure. When kanamycin-resistant transformants were selected at 47-degrees-C, the transformation efficiency of the plasmid bearing the TK101 gene was nine times higher than that of the plasmid encoding the wildtype enzyme. The difference in transformation efficiencies between the two plasmids was increased when transformants were selected at higher temperatures, reflecting the difference in thermostabilities of the respective kanamycin nucleotidyltransferases. We conclude that, even though the pUB110 enzyme is sufficiently active at 47-degrees-C to confer kanamycin resistance to B. stearothermophilus, the additional stability of the TK101 mutant is advantageous in transformation of B. stearothermophilus. The TK101 gene may also have broad utility as a marker for cloning vectors in other thermophiles.