NADH-dependent sulfite reductase (NADH-SIR) was purified to an electrophoretically homogeneous state from Escherichia coli harboring plasmid pTHS1, which has the S-adenosyl-L-methioninc : uroporphyrinogen III methyltransferase gene of Thiobacillus ferrooxidans. Purified NADH-S1R had an apparent molecular weight of 350,000, and showed absorption peaks characteristic of siroheme at 405 and 591 nm. The enzyme catalyzed the reduction of sulfite ion with NADH as an electron donor to hydrogen sulfide and NAD(+). However, NADPH could not be used as an electron donor. Preparations of both NADH-dependent sulite reductase from E. coli 32-15 (pTHS1) and iron-grown T. ferooxidans were specifically activated by molybdenum ion, suggesting that the properties of NADH-SiR from E. coli 32-15 (pTHS1) are similar to those of T. ferrooxidans.