In the present study, the initial rates of amino acid transport by isolated rat skeletal muscle plasma membrane vesicles were investigated. This approach facilitates the study of the transport of naturally occurring amino acids independent of the effects of cellular metabolism. Alanine and glutamine influxes were measured using a rapid filtration technique. Transport was examined in the presence and absence of Na and the properties of membranes from control, insulin-treated, or acutely exercised rats were studied. Both alanine and glutamine were transported by Na-dependent processes. The values for maximum rate of transport (Vmax) for Na-dependent alanine and glutamine transport were 203 and 224 pmol.mg-1.s-1, respectively. The K1/2 values were 2.9 mM alanine and 1.9 mM glutamine. The Vmax for Na-dependent alanine transport was increased by insulin treatment of the animal and by acute exercise. 2-(Methylamino)-isobutyric acid (MeAIB) partially inhibited the control Na-dependent alanine influx and completely inhibited the increase due to insulin or exercise treatment, indicating the importance of both system A and a non-system A, Na-dependent carrier for alanine transport. The Vmax for Na-dependent MeAIB uptake was also increased by insulin or exercise treatments of the rats. Unlike alanine, Na-dependent glutamine transport was not affected by insulin.