Bovine rumen fluid was fermented anaerobically over 48 h with cottonseed, corn, alfalfa, or a mixture of these substrates in anaerobic mineral buffer. Samples taken at different incubation times were derivatized with n-butanol and subjected to gas chromatography and mass spectroscopy. No unusual fermentation end-products from the cottonseed substrate were detected. Cottonseed supported rumen fermentation at levels comparable to those of the other substrates. Major components were usually found in the decreasing order of acetate, propionate, butyrate, and valerate, although acetate and propionate concentrations decreased late in the alfalfa and mixed-feed fermentations, eventually allowing butyrate concentrations to exceed those of propionate. As expected, lactate was produced in high concentrations when corn was fermented. The minor components 2-methylpropionate, 2- and 3-methylbutyrate, phenylacetate, phenylpropionate, and caproate also accumulated, with their relative concentrations varying with the substrate. Succinate was produced in substantial amounts only when corn and alfalfa were fermented; it did not accumulate when cottonseed was the substrate. Samples containing cottonseed were derivatized and subjected to reversed-phase high-performance liquid chromatography, revealing that gossypol concentrations did not change during fermentation.