The rate of total protein synthesis in isolated hepatocytes was determined. The incorporation of L-[3H]valine into protein is linear for at least two hours of incubation and is affected by the concentration of amino acids in the medium. Uptake of valine by hepatocytes from 1.5- and 18-month-old rats was identical and appears to occur by simple passive diffusion. Within five minutes, the specific activities of the intracellular and extracellular valine pools are equivalent. The specific activities of these pools are saturated by 1.6mM valine and remain constant for 60 minutes of incubation. The rates of protein synthesis by hepatocytes from 1- to 2-month-old rats is 96.8 pmoles of valine per minute per milligram protein. This is comparable to rates of protein synthesis reported for perfused liver and liver in vivo and is approximately 64% higher than the rate of protein synthesis by hepatocytes from 18-month-old rats.