A mixed culture of ruminal microorganisms was used to demonstrate that nickel (Ni) is incorporated into factor F430 and to determine the effects of monensin and formate on incorporation of Ni into factor F430. Ruminal microorganisms obtained from a semicontinuous culture were grown for 24 h in the presence of 63Ni and a 2 x 2 factorial arrangement of monensin (0 to 5 micrograms/ml) and formate (0 to 20 mM) treatments. Factor F430 was isolated and purified from the cultures by QAE-Sephadex A-25 column chromatography. The purified preparation contained 63Ni and exhibited a peak in absorbance at 430 nm. Methane production was decreased (P less than .01) 45% by monensin but was increased (P less than .01) 1.8-fold by formate. However, incorporation of 63Ni into factor F430, which is ubiquitous in methanogens and not found in other bacteria, did not parallel changes in methane production. Incorporation of 63Ni into factor F430 was decreased (P less than .01) 55% by monensin but was not affected (P greater than .05) by formate. In addition to its use for synthesis of urease and hydrogenase, Ni is involved in ruminal fermentation as a component of factor430.