Imipramine serum protein binding was measured by equilibrium dialysis in healthy subjects (88 women and 57 men; age 21 to 79 yr) who were relatively evenly distributed according to age, sex, smoking habits, and oral contraceptive use. Average free fraction was 10.9 +/- 1.4%. Interindividual variation in degree of binding was less than 100%, the free fraction varying from 8% to 14.7%. Women age 30 to 39 yr had significantly lower binding than all other female age groups and lower binding than men age 30 to 39 yr. Oral contraceptive use and smoking habits did not correlate to degree of binding. Serum concentrations of 12 proteins were measured in subjects with the highest binding (n = 17) and lower binding (n = 18). The concentration of orosomucoid, complement C3c, and apolipoprotein B was higher in the high-binding group than in the low-binding group. Since covariation among concentrations of these three proteins was modest, the data indicate a separate significance of the three proteins. The binding of 3H-imipramine did not correlate with the albumin concentration.