Chronic airway infection is a hallmark of cystic fibrosis (CF) and many CF patients are infected persistently by Staphylococcus aureus. Thymidine-dependent trimethoprim-sulfamethoxazole (SXT)-resistant S. aureus small-colony variants (SCVs), often in combination with isogenic normal S. aureus phenotypes, are highly prevalent and persistent in airway secretions of CF patients due to long-term SXT therapy (B. Kahl, M. Herrmann, A. S. Everding, H. G. Koch, K. Becker, E. Harms, R. A. Proctor, and G. Peters, J. Infect. Dis. 177:1023-1029, 1998). In this report, SCVs were compared to normal S. aureus by transcription analysis of important regulator (sigB, sarA, and agr) and virulence (alpha-hemolysin, hla, and protein A, spa) genes. Growth curve analyses revealed longer doubling times and lower final densities for SCVs than for normal strains. sigB activity was measured by transcription analysis of the sigB target gene asp23. For nearly all SCVs, expression of all regulators was decreased as assessed by asp23 reverse transcription-PCR for sigB and Northern analysis for sarA and agr. These results are in agreement with diminished hla signals in all SCVs and increased spa signals in 5 of 10 SCVs compared to the isogenic normal S. aureus. Both supplementation of SCVs with thymidine and activation of the agr quorum-sensing system by the supernatant of the isogenic normal strain reversed transcription to almost normal levels. In conclusion, multiple changes in growth characteristics and in regulator and virulence gene expression render SCVs less virulent and allow them to survive in the hostile environment present in the airways of CF patients, thereby illustrating adaptation of the bacteria during long-term persistence.