The effects of isoproterenol on the release of alanine during perfusion with pyruvate and valine were studied in perfused hindlimbs from rats that had been kept for 5 or 20 days at 4 degrees C. In hindlimbs perfused with Krebs bicarbonate buffer in a flow-through mode, the rate of release of alanine during perfusion with 2 mM pyruvate plus 5 mM valine was 250 nmol.min-1.leg-1, a rate that is comparable with that reported in hindlimbs perfused with complex medium. Neither the pyruvate-stimulated nor valine plus pyruvate-stimulated rates of release of alanine changed after 20 days of exposure to cold. Isoproterenol inhibited the release of alanine during perfusion with pyruvate, with valine, and with valine plus pyruvate in hindlimbs from a control group of rats. However, in hindlimbs from cold-exposed groups, isoproterenol failed to inhibit the release of alanine during perfusion with valine plus pyruvate and stimulated the release of alanine during perfusion with valine. Aminooxyacetate inhibited the effects of valine, pyruvate, and isoproterenol. The results obtained suggested that cold exposure decreases the responses to isoproterenol of the mechanism of alanine release and causes an increased supply of alanine to the liver.