A single bout of acute exercise increases hexokinase (HK) II mRNA and enzyme activity [R. M. O'Doherty, D. P. Bracy, H. Osawa, D. H. Wasserman, and D. K. Granner. Am. J. Physiol. 266 (Endocrinol. Metab. 29): E171-E178, 1994]. The present study addresses the mechanism of the increase in HK II mRNA. Male rats undertook a single bout of treadmill exercise and were then killed immediately or after a predetermined recovery period. The gastrocnemius/plantaris muscle complex, composed of mixed fiber types, was excised; the nuclei were isolated; and HK I, HK II, beta-actin, and alpha-tubulin gene transcription rates were measured. Genomic DNA and plasmid DNA were used as positive and negative controls, respectively. Immediately after the cessation of 30, 45, or 90 min of exercise, HK II gene transcription rates were 1.3 +/- 0.3-,2.9 +/- 0.3-, and 4.0 +/- 0.6-fold, respectively, above those of sedentary controls. The increases after 45 and 90 min of exercise were statistically significant (P < 0.01). One hour after the cessation of 30 min of exercise, HK II gene transcription was significantly increased (1.40 +/- 0.03-fold; P < 0.05). At all time points, transcription of the HK I, beta-actin, and alpha-tubulin genes was unchanged. We conclude that the exercise-induced increase in HK II gene transcription appears to play a major role in the increase of HK II mRNA and activity.