Activity of rifampin against 129 Streptococcus mitis isolates obtained from patients with hematologic cancer was investigated. One hundred twenty-five strains were susceptible to rifampin, and 4 were resistant (MIC = 32 to 64 mug/ml). Resistance to rifampin was related to mutations in the rpoB gene: His(526)Asn in three strains and His(526)Asp in one strain.