The effects of incorporation in the diet of 7% soya oil hydrolysate (SOH) on in vitro incubations of cellobiose + maltose, maize starch and casein by rumen microbes were studied using defaunated and refaunated sheep as rumen fluid donors. Feeding refaunated sheep the SOH supplemented diet lowered the protozoal numbers in the rumen from 1.61 10(6)/ml to 6.1 10(5)/ml. SOH addition reduced in vitro methane production, rather by a depletion of methanogens is than by a simple inhibition of their activity. This reduction seemed to be independent of protozoa depletion. With cellobiose-maltose and maize starch incubations, SOH supplementation increased molar proportion of propionate while acetate decreased. Both variations could be linked to the inhibition of methanogenesis. Volatile fatty acid production from casein was strongly reduced by SOH supplementation with or without protozoa in the rumen of the donors animals.