A specific, sensitive and accurate method for the determination of imipramine, desipramine and their d4 analogues in biological fluids using d8 analogues as internal standards using capillary gas chromatography-chemical ionization mass spectrometry was developed. Drug concentrations were measured by selected-ion monitoring of the quasi-molecular ions of imipramine and the trifluoro-acetyl derivative of desipramine. The coefficient of variation and relative error at a concentration of about 2 ng/ml in plasma were found less than 10% and 7%, respectively, for both drugs. No isotope effect was observed following the oral administration of an equimolar mixture of imipramine and [2H4]imipramine to a rat.