The interactions of the iron-molybdenum cofactor, FeMoco, isolated from acid-treated Azotobacter vinelandii molybdenum-iron protein (Av1) with EDTA and thiophenol in N-methylformamide solution have been reinvestigated. Our studies show that EDTA alone is sufficient to eliminate the EPR signal of dithionite-reduced FeMoco. Neither light/5-deazaflavin nor carbon monoxide are required, which implies that this EPR-silent form of FeMoco does not correspond to the EPR-silent, substrate-reducing state of Av1. As EDTA-treated FeMoco does not regain EPR activity on addition of sodium dithionite or thiophenol, it is apparently distinct from the EPR-silent form of either dye-oxidized FeMoco or dye-oxidized Av1. Thiophenol sharpens the EPR signal of dithionite-reduced FeMoco and shifts the g = 3.3 feature to g = 3.6. This shift is complete at 1:1 ratio of thiophenol/Mo atom, while the EDTA effect requires about 40 molecules/Mo atom. Thiophenol and EDTA probably affect different sites of FeMoco. The binding of either reactant does not affect the activity of FeMoco as measured by its ability to reconstitute extracts of A. vinelandii mutant UW45.