Amplification and sequencing of mycobacterial ribosomal RNA genes (16S rDNA) may permit the detection of growth-deficient species (i.e., those exhibiting no growth or those whose growth is delayed for more than 12 weeks). Of blood samples from 26 patients with AIDS and a liver sample from one additional AIDS patient, three samples (two of blood and the one of liver) were positive by polymerase chain reaction only; cultures of these three samples remained negative for more than 12 weeks. Analysis of amplified 16S rDNA from blood revealed a sequence characteristic of Mycobacterium genavense in the first case, in which one of many previous blood cultures had also been positive for M. genavense. The sequences found in the second and third cases were characteristic of Mycobacterium avium. The sample from the second patient was a liver biopsy specimen in which acid-fast bacilli were visualized; the culture of this specimen yielded M. avium after 7 months. The third sample was a blood sample from a patient in whom a relapse of treated M. avium infection was suspected. These results indicate that amplification and sequencing of mycobacterial 16S rDNA may permit early diagnosis and provide a rationale for treatment of infections due to growth-deficient mycobacteria.