A mixed methanogenic culture was highly enriched in a growth medium containing propionate as the sole organic carbon and energy source. With this culture, the pathways of propionate degradation were studied by use of 14C-radiotracers. Propionate was first metabolized to acetate, carbon dioxide, and hydrogen by nonmethanogenic organisms. Formate was not excreted. The carbon dioxide originated exclusively from the carboxyl group of propionate, whereas both [2-14C]- and [3-14C]propionate lead to the production of radioactive acetate. The methyl and carboxyl groups of the acetate produced were equally labeled, regardless of whether [2-14C]- or [3-14C]propionate was used. These observations suggest that in the culture, propionate was degraded through a randomizing pathway.