We have previously shown that 12 wk of endurance training reduces the rate of glucose appearance (Ra) during submaximal exercise (Coggan, A. R., W. M. Kohrt, R. J. Spina, D. M. Bier, and J. O. Holloszy. J. Appl. Physiol. 68: 990-996, 1990). The purpose of the present study was to examine the time course of and relationship between training-induced alterations in glucose kinetics and endocrine responses during prolonged exercise. Accordingly, seven men were studied during 2 h of cycle ergometer exercise at approximately 60% of pretraining peak oxygen uptake on three occasions: before, after 10 days, and after 12 wk of endurance training. Ra was determined using a primed, continuous infusion of [6,6-2H]glucose. Ten days of training reduced mean Ra during exercise from 36.9 +/- 3.3 (SE) to 28.5 +/- 3.4 mumol.min-1.kg-1 (P < 0.001). Exercise-induced changes in insulin, C-peptide, glucagon, norepinephrine, and epinephrine were also significantly blunted. After 12 wk of training, Ra during exercise was further reduced to 21.5 +/- 3.1 mumol.min-1.kg-1 (P < 0.001 vs. 10 days), but hormone concentrations were not significantly different from 10-day values. The lower glucose Ra during exercise after short-term (10 days) training is accompanied by, and may be due to, altered plasma concentrations of the major glucoregulatory hormones. However, other adaptations must be responsible for the further reduction in Ra with more prolonged training.