The importance of variation in plasma binding in the uncertain correlation between total plasma concentration and the antidepressive effect of amitriptyline (AT) and its active metabolite nortriptyline (NT) was examined. Plasma binding of AT and NT in 131 plasma samples from 87 patients was analyzed by equilibrium dialysis at 37 degrees for 3 hr. There was a twofold variation in percent unbound AT and NT (range 3.5% to 8.6% and 5.4% to 11.3%) and there was no correlation between percent and unbound drug and total drug concentration (range 54 to 6910 nmol/l and 77 to 3420 nmol/l for AT ant NT). The correlation coefficient relating unbound drug concentration of AT and NT to total concentration in plasma was 0.99 for the whole group and 0.16 for the NT therapy control patients, with total plasma concentrations within the therapeutic range. At therapeutic concentration AT was 66.6% and 63.5% bound to purified isolated orosomucoid (alpha 1-acid glycoprotein) and albumin, at physiologic concentrations. The binding to isolated lipoproteins was not examined, but no correlation was found between percent unbound AT and NT and plasma concentration of triglycerides, cholesterol, or orosomucoid.