Tricyclic antidepressants, especially amitriptyline (AMI), are widely used by patients who require tests of autonomic function. AMI effects autonomic function, but its quantitative and differential effects are not known. We prospectively evaluated the effect of AMI on sudomotor (M3 receptor), cardiovagal (M2) and adrenergic functions in 6 subjects, aged 20-40 years before, during, and 48 hours after AMI 75 mg/d. M3 receptor function was evaluated using QSART (quantitative sudomotor axon reflex test) recordings from four sites, M2 receptor function from cardiovagal studies [HR response to deep breathing (HRDB) and the Valsalva ratio (VR)], and adrenergic function from an analysis of beat-to-beat BP responses to tilt and the Valsalva maneuver. Plasma AMI was determined from blood samples obtained within 2 hours of autonomic testing. QSART volume was reduced by 47% by AMI (P = 0.01), and recovered to 81% of baseline following a 48-hour washout. HRDB was unaffected by AMI (P > 0.05). VR was increased by 10% (NS) with AMI and by 14% (P < 0.05) with washout. The percent change in hemodynamic parameters by AMI were: orthostatic reduction in SBP, 62% (NS); orthostatic HR increment, 66%, (P = 0.03); and phase IIe SBP decrement, 665%, (P = 0.02). We conclude that AMI in the moderate doses used, resulted in greater inhibition of M3 than M2 receptors. The BP, HR, and beat-to-beat BP alterations likely reflect adrenergic inhibition resulting in a reduction in effective plasma volume. A washout of 48 hours is adequate for muscarinic but not adrenoreceptors.