Rapid unidirectional transport (15 sec) of L-serine and 2-methylaminoisobutyric acid (MeAIB) was studied in the isolated perfused rat pancreas using a dual-tracer dilution technique. Time-course experiments in the presence of normal cation gradients revealed a time-dependent transstimulation of L-serine influx and transinhibition of MeAIB influx. Transport of the model nonmetabolized System A analog MeAIB was Na+ dependent and significantly inhibited during perfusion with 1 mM ouabain. Although transport of L-serine was largely Na+ independent, ouabain caused a time-dependent inhibition of transport. Influx of both amino acids appeared to be inhibited by the ionophore monensin but unaffected by a lowered extracellular potassium concentration. Removal of extracellular calcium had no effect on influx of the natural substrate L-serine, whereas stimulation of transport by exogenous insulin (100 microU/ml) was entirely dependent upon extracellular calcium and unaffected by ouabain. Paradoxically, exogenous insulin had no effect on the time-course of MeAIB influx. The characteristics of L-serine influx described in earlier studies together with our present findings suggest that insulin may modulate the activity of System asc in the exocrine pancreatic epithelium by a calcium-dependent mechanism.