Leather samples were prepared and characterized as 'in house' matrix standards for the determination of fat. The Soxhlet standard method was used to establish the reference fat content in every standard sample. Sample homogeneity and stability were examined under specific storing conditions. The materials were subsequently used as matrix standards for the determination of fat in leather samples, using supercritical fluid extraction (SFE) with on-line piezoelectric detection. Real samples were weighed in the extraction SFE thimble, previously loaded with 1 g of diatomaceous earth. A temperature of 45 degrees C and a CO2 fluid density of 0.85 g ml-1 were used for extraction. The linear calibration range thus achieved was 0.001-0.040% m/m total fat (related to the weight of the leather) and the relative standard deviation +/- 3% (n = 11; P = 0.05). The results were compared with those obtained with the Soxhlet method and no significant differences were found.