One important aspect of macrophage function is the production of inflammatory and anti-inflammatory cytokines, which in turn affect the survival of intracellular organisms such as mycobacteria.To determine the relationship between phagocytosis of mycobacteria and expression of intracellular cytokines.Phagocytosis and cytokine production were studied simultaneously within human monocyte-derived macrophages (MDMs) from healthy donors using fluorescent labelling of M. bovis BCG and flow cytometry.At a range of infection ratios (5:1, 1:1, 0.2:1) TNF- alpha, IL-10, IL-6 and IL-12 were all produced in a dose-dependent manner. At an infection ratio representative of the in vivo situation (1:1), cytokine production was induced in both MDMs containing intracellular M. bovis BCG and in uninfected bystander MDMs. Phagocytosis increased over time, but there was considerable donor variation: the proportions of cells containing one or more mycobacterium were 15.4+/-14.8% (mean+/-SD) at 4 h and 32.7+/-21.1% at 24 h (n=19). Analysis of cytokine production by MDMs not containing mycobacteria (bystander cells) at 4 h revealed that these uninfected cells produced 79+/-6.6% of the TNF- alpha, 53.9+/-40.0% of the IL-10 and 64.2+/-12.4% of the IL-12. By 20 h these proportions had decreased to 57+/-13.5%, 30.9+/-7.4% and 45.5+/-13.3% respectively.Both infected and bystander MDMs can be stimulated to produce cytokines in response to M. bovis BCG, indicating that the ability of MDMs to produce cytokines is not necessarily dependent on the ability to phagocytose mycobacteria.