This paper compares the blood-to-serum distribution (B/S ratio) of 3,4-methylenedioxymethamphetamine (MDMA) and its major metabolite 3,4-methylenedioxyamphetamine (MDA). B/S ratios were determined by liquid chromatography-tandem mass spectrometry analysis following liquid-liquid extraction as a function of the hematocrit value (experimental specimens) and in blood and corresponding serum samples (n = 63) from 16 healthy volunteers participating in a controlled driving experiment (authentic specimens). A regression analysis to calculate the B/S ratio was performed followed by an analysis of covariances (ANCOVA). A linear relationship between the hematocrit value and the B/S ratio of both MDMA and MDA could be established from the experimental data. For MDMA, the regressions provided mean B/S ratios of 1.22 and 1.26 for the experimental setting and the authentic samples, respectively. For MDA, the analysis determined slopes of 1.15 and 1.27 for the experimental setting and field study, respectively. ANCOVA revealed that the method of determination (experimental vs. authentic specimens) did not influence the resulting slopes. A conversion factor of 0.80 may give an adequate estimate to derive the serum concentration for MDMA if only the concentration in whole blood is known, whereas such a definitive factor could not be established for MDA because of its very low levels in authentic samples.