The immunologically active vitamin retinoic acid (RA) was tested for the ability to increase the resistance of cultured human macrophages (MP) to experimental infection with virulent Mycobacterium tuberculosis Erdman (tubercle bacilli [TB]). It was added to MP in various concentrations and addition regimens. Protection against TB was measured by counting live TB (CFU) in lysates of samples of MP taken at 0, 4, and 7 days after MP infection. RA was protective when added after infection at the pharmacologic concentration of 10(-5) M and when added before infection at the physiologic concentration of 10(-7) M. The protection lengthened intracellular generation times for TB, occasionally caused bacteriostasis, and regularly kept CFU counts at 7 days (end of the period of infection) 1 to 2 log10 CFU below control values. Significant protection was seen in a series of 16 experiments with MP from seven different donors, but the degree of protection varied considerably. The protection depended partly on and was inversely proportional to concentrations of a serum substitute or autologous serum used as a supplement in the RPMI 1640 MP culture medium. It was strongest at concentrations of serum below 1%. RA at concentrations used in the MP cultures did not inhibit TB in the absence of MP. These results suggest that RA (vitamin A), like vitamin D, may have some immunoprotective role against human tuberculosis, as historically intimated by the regular use of vitamin A- and D-rich cod liver oil for the treatment of tuberculosis before the introduction of modern chemotherapy.