This study was designed to compare the activity of skeletal muscle carnitine palmitoyltransferase I (CPT I) in trained and inactive men ( n = 14) and women ( n = 12). CPT I activity was measured in intact mitochondria, isolated from needle biopsy vastus lateralis muscle samples (∼60 mg). The variability of CPT I activity determined on two biopsy samples from the same leg on the same day was 4.4, whereas it was 7.0% on two biopsy samples from the same leg on different days. The method was sensitive to the CPT I inhibitor malonyl-CoA (88% inhibition) and therefore specific for CPT I activity. The mean CPT I activity for all 26 subjects was 141.1 ± 10.6 μmol ⋅ min−1 ⋅ kg wet muscle (wm)−1 and was not different when all men vs. all women (140.5 ± 15.7 and 142.2 ± 14.5 μmol ⋅ min−1 ⋅ kg wm−1, respectively) were compared. However, CPT I activity was significantly higher in trained vs. inactive subjects for both men (176.2 ± 21.1 vs. 104.1 ± 13.6 μmol ⋅ min−1 ⋅ kg wm−1) and women (167.6 ± 14.1 vs. 91.2 ± 9.5 μmol ⋅ min−1 ⋅ kg wm−1). CPT I activity was also significantly correlated with citrate synthase activity (all subjects, r = 0.76) and maximal oxygen consumption expressed in milliliters per kilogram per minute (all subjects, r = 0.69). The results of this study suggest that CPT I activity can be accurately and reliably measured in intact mitochondria isolated from human muscle biopsy samples. CPT I activity was not affected by gender, and higher activities in aerobically trained subjects appeared to be the result of increased mitochondrial content in both men and women.