.-The resting content and use of myocellular triacylglycerol (MCTG) during 90 min of submaximal exercise [60% of peak oxygen uptake (V O2 peak)] were studied in 21 eumenorrheic female and 21 male subjects at different training levels [untrained (UT), moderately trained (MT), and endurance trained (END)]. Males and females were matched according to their V O2 peak expressed relative to lean body mass, physical activity level, and training history. All subjects ingested the same controlled diet for 8 days, and all females were tested in the midfollicular phase of the menstrual cycle. Resting MCTG, measured with the muscle biopsy technique, averaged 48.4 Ϯ 4.2, 48.5 Ϯ 8.4, and 52.2 Ϯ 5.8 mmol/kg dry wt in UT, MT, and END females, respectively, and 34.1 Ϯ 4.9, 31.6 Ϯ 3.3, and 38.4 Ϯ 3.0 mmol/kg dry wt in UT, MT, and END males, respectively (P Ͻ 0.001, females vs. males in all groups). Exercise decreased MCTG content in the female subjects by an average of 25%, regardless of training status, whereas in the male groups MCTG content was unaffected by exercise. The arterial plasma insulin concentration was higher (P Ͻ 0.05) and the arterial plasma epinephrine concentration was lower (P Ͻ 0.05) in the females than in the males at rest and during exercise. MCTG use was correlated to the resting concentration of MCTG (P Ͻ 0.001). We conclude that resting content and use of MCTG during exercise are related to gender and furthermore are independent of training status. muscle substrate; training; triglycerides IT HAS PREVIOUSLY BEEN SHOWN (25) that myocellular triacylglycerol (MCTG) is utilized during the postexercise period. MCTG stores also represent a potentially large energy source during exercise. However, the extent to which MCTG is used during exercise and the possible existence of differences in its use between trained and untrained (UT) subjects are still under debate. In studies (27, 37) using stable isotope techniques combined with indirect calorimetry, it was estimated that MCTG accounted for 20-25% of the oxidative metabolism during submaximal exercise. However, when direct measurements of MCTG concentration in muscle biopsies have been used, some studies (4, 19, 34) have found a decrease in MCTG concentration during submaximal exercise, whereas others (1, 21, 22, 25, 44) have observed no change. In all of the above-mentioned studies, only male subjects have participated. Thus it is unknown whether gender differences exist in the utilization of MCTG during exercise. Some studies (18,45,46) have shown that females utilize lipids to a greater extent than males during submaximal exercise, but to our knowledge it has not been investigated whether this increased lipid utilization in females is primarily from MCTG or other lipid sources. Other studies (3, 6, 32) have not been able to find gender differences in lipid utilization during exercise. This could be due to differences in training status and exercise mode in the experimental designs. The aim of the present study was therefore to evaluate the contribution of MCT...