The effect of two new macrolides, azithromycin and roxithromycin used either alone or in combination with amikacin rifabutine and 1.25 (HO)2 vitamin D3 was examined in vitro. Macrophage monolayers infected with M. avium complex or M. xenopi were treated with antibiotics or 1.25 (OH)2 vitamin D3 by using different protocols: antibiotics or 1.25 (OH)2 vitamin D3 was added to the macrophage monolayers immediately after infection and released by washing out after 24 h; antibiotics or 1.25 (OH)2 vitamin D3 was replenished daily for 4 days; or infected macrophage monolayers were treated with antibiotics plus vitamin D3 for 4 consecutive days. Treatment for 24 h resulted in an inhibition of growth. After treating the macrophage monolayers with the four antibiotics alone or in combination for 4 consecutive days intracellular killing of mycobacteria was observed and the combinations were significantly more lethal than antibiotics alone. The mycobactericidal effect was enhanced when vitamin D3 was added to the culture.