Peptide metabolism by rumen microorganisms was investigated by gel filtration using Sephadex G-25 with water as eluant. Protein hydrolysates containing a mixture of peptides were used to calibrate the column. Total peptide in each fraction was estimated from its A206, and free peptide amino groups were analysed by fluorescamine, thus enabling the average peptide Mr to be calculated. Three different protein hydrolysates produced similar, nearly linear, relations between log Mr and elution volume for peptides between 300 and 2000 Da. Trypticase, a pancreatic hydrolysate of casein, was metabolised rapidly in rumen fluid in vitro. Hydrolysis appeared to be complete after 6 h, leaving a small residual peptide concentration which persisted up to 24 h, equivalent to about 15% of the original peptide concentration of 2 g litre−1. Residual peptides from casein hydrolysis were 0.05 g litre−1 at 24 h. Peptides accumulated in rumen fluid of sheep receiving dietary ionophores. Two hours after feeding, the accumulation with monensin appeared to be of peptides of a wide Mr range, while tetronasin caused an accumulation mainly of smaller, <570 Da, peptides. Treatment of Trypticase with acetic anhydride afforded 76% protection of its peptides from degradation to ammonia in a 6-h incubation. When Trypticase was fractionated by gel filtration then acetylated, none of the fractions was protected significantly better than others.