Effects of varying protein level on hepatic utilization of serine, threonine and glycine were examined by measurements of metabolic fluxes across the liver. Feeding a high protein (HP) diet markedly enhanced hepatic extraction of serine, threonine and glycine, in parallel to alanine. After 20 hours starvation, activity of alanine aminotransferase and serine dehydratase still reflected the induction of these enzymes in fed rats. Thus, in starved rats previously adapted to HP diets, hepatic uptake of serine, threonine and glycine remained very efficient. With a normal diet, gluconeogenesis from alanine may be very active during starvation, in contrast to serine. The present results suggest that serine, and, to a lesser extent glycine, are very efficient glucogenic substrates with HP diets. The serine aminotransferase pathway might be important in rats fed HP diets, particularly for utilization of serine synthesized from glycine in mitochondria. With HP diets, the drop in hepatic alanine, serine and threonine suggest that transport across the plasma membrane might limit their utilization.