A bacteriophage (phiYS40) infectious to an extreme thermophile, Thermus thermophilus HB8, was isolated and characterized. phiYS40 grows over the temperature range of 56 to 78 C, and the optimum growth temperature is about 65 C. The phage had a latent period of 80 min and a burst size of about 80 at 65 C. The phage has a hexagonal head 0.125 mum in diameter, a tail 0.178 mum long and 0.027 mum wide, a base plate and tail fibers. The phage is thermostable in broth but rather unstable in a buffer containing 10 mM Tris, 10 mM MgCl2, pH 7.5. The addition of Casamino Acids (1 percent), polypeptone (0.8 percent), yeast extract (0.4 percent), NaCl (0.1 M) or spermidine (1 mM) to the buffer restores the thermostability of phiYS40 to the same degree as in broth. The phage is also thermostable in water of the hot spring from which this phage was isolated. The nucleic acid of PhiYS40 is a double-stranded DNA and has a molecular weight of 1.36 X 10-8. The guanine plus cytosine content of the DNA was determined to be about 35 percent from chemical determinations, buoyant density (1.693 g/cm-3 in CsCl), and melting temperature (83.5 C in 0.15 M NaCl plus 0.015 M sodium citrate).