The time-kinetics of the intracellular bioactivity and intracellular post-antibiotic effect (PAE) of rifabutin and sparfloxacin against Staphylococcus aureus and Mycobacterium tuberculosis, grown in human monocytes, were evaluated. Intracellular bactericidal activity against staphylococci was shown in the presence of extracellular drug concentrations equal or superior to 1/10 plasma Cmax. The bactericidal activity of rifabutin was dependent on both its extracellular concentrations and the exposure time. In contrast, the pattern of the intracellular activity of sparfloxacin was characterized by a minimal concentration dependent killing. Both antibiotics (from 1/10 to the expected lung Cmax) showed intracellular bioactivity against M. tuberculosis H37Ra and H37Rv strains. A long intracellular PAE on staphylococci (>4 hours) was demonstrated when drugs were removed from the infected monocytes after 1 h treatment. Our findings suggest that rifabutin and sparfloxacin may be useful in the treatment of lower respiratory tract infections due to intracellular pathogens.