Whole-cell suspensions of Enterobacter cloacae SLD1a-1 produced dimethylselenide (DMSe) from selenate, selenite, elemental selenium, dimethylselenone, seleno-DL-methionine, 6-selenoinosine, and 6-selenopurine. Cell-free extracts of the bacterium produced the formation of DMSe from organic selenium compounds, including dimethylselenone, dimethylselenoniopropionate, seleno-DL-methionine, seleno-DL-ethionine, and 6-selenoguanosine. The highest rate of DMSe production occurred from whole-cell suspensions and cell-free extracts containing dimethylselenone. DMSe was also produced by cell-free extracts containing selenite or elemental selenium and methylcobalamin. Cell-free extracts did not produce DMSe from inorganic selenium when S-adenosyl-L-methionine was present. Additionally, DL-homocysteine and L-methionine were found to inhibit selenium volatilization. These findings suggest the formation of DMSe from inorganic selenium occurs through the transfer of a methyl group from methylcobalamin.