Pyrazinamide (PZA) is believed to be mycobactericidal in vivo. Because it is ineffective at neutral pH in vitro, it is thought to owe its in vivo activity at least partly to acting upon tubercle bacilli (TB) in the helpfully low pH of macrophage (MP) phagolysosomes. However, when it was tested in TB-infected cultured human MP, it was not bactericidal and was able only to slow intra-MP bacillary growth. Recent evidence has suggested that human MP need hormonal support from certain vitamin D metabolites to resist TB. This support was not provided in the culture medium of the earlier experiments in which the PZA was relatively ineffective. Here, PZA has been retested in MP cultured in medium supplemented with the hormonally active metabolite of vitamin D, 1,25(OH)2-vitamin D3 (1,25D3). The MP were infected with virulent TB and incubated in various concentrations of PZA. 1,25D3 was added to postinfection medium at 4 micrograms/ml. Inhibition or killing of intracellular TB was quantitated by counts of culturable TB from samples of lysed MP taken at zero, 4, and 7 days after MP infection. Previous evidence for the protectiveness of 1,25D3 alone for human MP against TB was confirmed. The weak inhibition of TB in MP by PZA alone also was confirmed. The two used together synergized to decrease concentrations of PZA which were inhibitory and to switch the action of PZA from weakly inhibitory to bacteriostatic or mildly bactericidal. 1,25D3 had no direct anti-TB effect, and it did not synergize with PZA in the absence of MP, as determined with acidified bacteriologic culture medium in the BACTEC radiometric system.(ABSTRACT TRUNCATED AT 250 WORDS)