The influence of peptides, amino acids and ammonia on the growth and activity of rumen microorganisms was determined in vivo by infusing solutions of peptides, amino acids or ammonia into the rumen and in vitro by adding peptides, amino acids or ammonium sulphate to a nitrogen (N)-free basal growth medium. Four adult sheep were fed grass hay (2% N) at 32 g organic matter (OM) per kg body weight, divided into equal meals at 08:00 and 16:00 h. Solutions of enzymic casein hydrolysate (peptides), casein acid hydrolysate + tryptophan, methionine and cysteine (amino acids), and urea were infused into the rumen via a rumen cannula to provide 150 mg N per kg body weight daily. Each treatment period was 21 days, and animals received the infusions or an infusion of the same volume of water (20 ml h) in a 4 × 4 Latin square design. Rumen pH, numbers of total viable bacteria, cellulolytic bacteria and ciliate protozoa, and volatile fatty acid concentrations were unaffected by the treatments (P > 0.05), except for the higher and branched-chain acid concentrations which increased (P <0.05) when peptides and amino acids were infused. Rumen ammonia concentration increased with all N infusions (P <0.05). The degradation of OM of grass hay incubated in nylon bags in the rumen was unaffected (P > 0.05) by the N infusions. The digestibility of the hay was low (58.6%) and unaffected by the infusions. The flow of microbial protein from the rumen, calculated from the urinary excretion of purine derivatives, was also unaffected (P > 0.05). In pure cultures, peptides and amino acids stimulated the growth rate of the cellulolytic bacteria, Ruminococcus albus SY3, Ruminococcus flavefaciens 17 and Fibrobacter succinogenes BL2 on cellobiose but not cellulose (Avicel). Peptides showed no benefit over amino acids with the celluloytic bacteria, in contrast to the non-cellulolytic species, Megasphaera elsdenii, Prevotella ruminicola, and Selenomonas ruminantium, whose growth rates were stimulated by more than 70% by peptides in comparison with amino acids. It was concluded that fermentation by rumen bacteria is not limited by the availability of peptides or amino acids when ammonia is available and the growth rate is limited by a slowly degradable energy source.