The characteristics of the accumulation of 2 mg/L [(14)C]rifampicin by wild-type strains of Mycobacterium aurum (A(+)), Mycobacterium smegmatis (mc(2)155) and Mycobacterium tuberculosis (H37Rv) were determined. After 10 min exposure M. aurum had accumulated 220 ng rifampicin/mg cells, M. smegmatis had accumulated 120 ng rifampicin/mg cells and M. tuberculosis had accumulated 154 ng rifampicin/mg cells. A steady-state concentration (SSC) of rifampicin was accumulated rapidly by M. aurum and M. tuberculosis within minutes of drug exposure, unlike M. smegmatis, which accumulated rifampicin more slowly. With an increase in the concentration of rifampicin from 0.12 mg/L to 2 mg/L there was an increase in the concentration of rifampicin accumulated by M. tuberculosis, with no detectable loss of viability over the 20 min of the accumulation experiment. With an increase in temperature there was also an increase in the concentration of rifampicin accumulated by M. tuberculosis; between 15 and 30 degrees C the increase was linear. For all three species sub-inhibitory concentrations of ethambutol increased the concentration of rifampicin accumulated. However, both growth and accumulation of rifampicin were lower in the presence of 0.05% Tween 80. Accumulation of rifampicin by M. smegmatis was unaffected by the presence of the proton motive force inhibitor, 2,4-dinitrophenol (1 mM), whether added before or after the addition of rifampicin to the mycobacterial culture. For all three species, the Gram-positive bacterial efflux inhibitor reserpine (20 mg/L) slightly increased the SSC of rifampicin, but the increase was not statistically significant. Addition of glucose to energize a putative efflux pump had little effect on the accumulation of rifampicin in the presence or absence of reserpine for M. tuberculosis; however, for M. aurum and M. smegmatis the reserpine effect was abolished by the addition of glucose. These data suggest that rifampicin may be removed from wild-type mycobacteria by efflux, but that the pump(s) is expressed at low level.