Exposure of mice to a single or multiple low doses of ultraviolet radiation (UVR) decreases the induction of the delayed-type hypersensitivity (DTH) response to Mycobacterium bovis BCG and Mycobacterium lepraemurium (MLM) and impairs the clearance of bacteria from the lymphoid organs. This study is an attempt to address the mechanism by which UV radiation impairs the clearance of bacteria from the lymphoid organs by determining whether alterations in macrophage function such as ingestion and intracellular killing of mycobacteria or production of reactive nitrogen intermediates might be responsible for these effects. BALB/c or C3H/HeN mice were exposed to a single dose of UVB (280–320 nm) radiation ranging from 0.35 to 45 kJ/m², and at regular intervals after irradiation, the peritoneal and splenic macrophages were collected, cultured, and infected with live BCG or MLM. Phagocytosis was assessed at 6 h by counting the number of acid-fast bacteria per macrophage after Ziehl-Neelsen staining. The rate of intracellular killing was assessed by lysing the macrophages at 6, 12, 24, and 48 h after BCG infection, plating the suspension on 7H11 agar, and counting the number of colony-forming units 21 days later. Similarly, the nitric oxide production, as measured by nitrite, by macrophages obtained from UVB-irradiated and nonirradiated mice in response to BCG was assessed. There was a significant reduction in the uptake of organisms by both peritoneal and splenic macrophages collected from UV-irradiated mice. The intracellular killing of organisms was also significantly reduced, as was the production of nitric oxide by peritoneal macrophages infected with BCG in vitro. These results indicate that UVR affects macrophage functions and are consistent with our hypothesis that impaired clearance of bacteria in vivo results from an alteration in macrophage function. J. Leukoc. Biol. 57: 883–890; 1995.