Cultured rat hepatoma cells accumulate 2-aminoisobutyrate to high concentrations by a transport mechanism probably of the A type mediation. 2. Transport is enhanced by the presence of serum. When cells are deprived of serum the rate of transport declines over a period of hours; conversely addition of serum leads over a period of hours to increase in transport activity. In the presence of serum the apparent Km for aminoisobutyrate uptake is about 8 mM. In cells deprived of serum the Km is much higher. 3. Addition of insulin produces both an immediate increase in the rate of aminoisobutyrate uptake and a time-dependent rise. 4. The presence of alanine diminished aminoisobutyrate uptake in a concentration-dependent fashion. Competition is seen both in the presence and absence of serum but not when cells are incubated at 4 degrees C. 5. Preincubation with alanine for 1 h also diminishes aminoisobutyrate uptake when the alanine is removed. Cells take a period of several hours to recover from the depression of transport induced by alanine. 6. Transport of aminoisobutyrate rapidly declines in the presence of cycloheximide. Actinomycin had no effect for at least 8 h.