Two methods for the determination of fentanyl at subnanogram concentrations in whole blood have been developed and evaluated. The initial screening was by gas chromatography with nitrogen sensitive detection (GC/NPD) in a splitless injection onto two fused-silica, 0.32-mm i.d. capillary columns (5% and 50% phenyl methyl silicone). Confirmation was by gas chromatography/mass spectrometry (GC/MS) using selected ion monitoring of a splitless injection onto a 0.1-mm i.d., 0.34-microns 5% phenyl methyl silicone capillary column. The methods were studied at fentanyl concentrations over the range 0.05 to 5.0 ng/mL using 2 mL of blood. The detection limits were set at 0.10 ng/mL for GC/NPD and 0.05 ng/mL for GC/MS. The overall recovery of fentanyl was found to be greater than 75% over the range of 0.25 to 2.5 ng/mL. The within-run precision determined at fentanyl concentrations of 0.25 and 1.0 ng/mL showed coefficients of variation ranging from 8.7 to 14.8%. The between-run precision determined at concentrations of 0.4 and 0.8 ng/mL showed coefficients of variation ranging from 3.3 to 11.6%. The blood calibration curves in the range of 0.25 to 2.5 ng/mL monitored over a 3-month period showed a mean correlation coefficient of 0.99 for both the GC/NPD and GC/MS methods.