Supplementation with CaCl2 . 2H(2)O (40 mg l(-1)) or CuSO4 . 5H(2)O (10 mg l(-1)) improved vancomycin production by Amycolatopsis orientalis by 12 and 11%, respectively; used in combination, they acted synergistically. Ca2+ decreased the intracellular concentration of vancomycin 36%, whereas Cu2+ increased the intracellular activity of TDP-glucose:aglycosylvancomycin glucosyltransferase 3 times more than control. Ca2+ probably works by altering the permeability of cells to vancomycin, whereas Cu2+ increases the activity of an enzyme responsible for vancomycin biosynthesis.