Five rumen-fistulated wethers were dosed intraruminally with a single dose of 75Se-selenomethionine, and the rumen liquor was collected after various time intervals and separated into bacterial, cell-free, and protozoa plus plant material fractions. Maximum 75Se activity in rumen fluid was observed 2 h after labelled selenomethionine was administered. At 6 h post-dosing, 50% of the rumen liquor label was in the bacterial fraction, decreasing to 20% at 96 h. The majority of bacterial 75Se activity was found to be protein-bound. In a second experiment, five wethers were given 75Se-selenomethionine intraruminally, and 2 h later ruminal bacteria were isolated for identification of selenocompounds in the bacterial protein. Paper chromatographic separation of protein enzymatic hydrolysate showed the presence of selenocystine, selenomethionine, elemental selenium, and 40–50% 75Se activity as unidentifiable components. In an in vitro study, 75Se-selenomethionine was incubated with a rumen bacterial fraction, and ion-exchange and paper chromatography were used to identify selenocompounds formed. The results indicated that 75Se-selenomethionine was metabolized by the bacteria to 75Se-selenocystine, with both selenoamino acids incorporated into bacterial protein. With ion-exchange chromatography, unless carrier selenomethionine was added, 75Se-selenomethionine was degraded and a high proportion of 75Se activity remained on the column.