The effect of exposure of chick embryo cells to increasing concentrations of Na+ in the culture medium on the subsequent amino acid transport as determined at physiological osmolarity was investigated in detail. It was found that the hyperosmolar treatment stimulated amino acid transport in a dose-dependent manner up to 200 mM Na+. Changes were measurable as early as 1 h after altering Na+ and reached a maximum after 4 h, remaining constant thereafter. The maintenance of this effect required continuous exposure of the cell to high Na+ in the culture medium. Hyperosmolarity-mediated increases in amino acid transport activity by system A have been detected with L-proline and L-alanine. Transport activities of systems ASC and L did not change appreciably after exposure of the cells to high Na+. Inhibition of protein synthesis by cycloheximide or RNA synthesis by actinomycin D (actD) prevented these uptake changes. Kinetic analysis indicated that the stimulation of the activity of transport system A by high Na+ treatment occurred through a mechanism affecting Vmax rather than Km.