Many investigators assume the protein concentration and colloid osmotic pressure of interstitial fluid and lymph to be identical even after the lymph has passed through a lymph node. We quantitated the degree of modification of lymph by the dog popliteal lymph node by perfusing isolated lymph nodes in situ at physiological flow rates with homologous plasma or plasma diluted to low protein concentration. This enabled us to compare directly prenodal and postnodal lymph flows and protein concentrations. When undiluted plasma was infused into the node, fluid filtered from the blood into the lymph, diluting the lymph. When diluted plasma was infused, fluid was absorbed from the lymph, concentrating the lymph. Nearly all (98%) of the change in lymph protein concentration could be explained by transfer of protein-free fluid either into or out of the lymph. However, when the nodes were perfused with lymph having a colloid osmotic pressure that exactly balanced the hydrostatic and osmotic forces acting across the lymph node blood-lymph barrier, the lymph was not modified during nodal transit. This "equilibrium colloid osmotic pressure" averaged 60% of that of plasma. The concentrating-diluting mechanism became more significant as the perfusion rate decreased and/or as the colloid osmotic pressure of the afferent lymph was made progressively greater than or less than the equilibrium colloid osmotic pressure. We conclude that lymph nodes modify lymph protein concentration and colloid osmotic pressure except when these are already at equilibrium values for given lymph node conditions. Therefore, the assumption that postnodal lymph is representative of interstitial fluid, especially at low but still physiological lymph flows, is likely to be incorrect.