The metabolic pattern of cardioactive and inactive conjugated metabolites of digitoxin on maintenance (9 patients) and after a single 0.6-mg dose (5 patients) was studied in patients with normal renal and hepatic function. Serum samples were obtained 24 hr after the last dose, and urine was collected over 24 hr. The extent of conjugation to glucuronic and sulfuric acid was 35.0% (SD, 17.4) in whole serum and 31.6% (SD, 19.3) in urine samples. Unchanged digitoxin was the main cardioactive substance found both in serum and in urine (89.7% and 87.0%) in the steady-state group. All known cardioactive metabolites were present; digoxin represented less than 1%. All active metabolites were conjugated to glucuronic/sulfuric acid. Serum and urine patterns of metabolites were quite similar, Hydrolysis and conjugation appeared to be more important pathways than hydroxylation. Unchanged digitoxin was the most important cardioactive substance in serum and urine (80.4% and 56.5%) in the single-dose group. Digoxin was the main cardioactive metabolite (12.5% in serum and 25.5% in urine). All active metabolites were conjugated. Hydroxylation, hydrolysis, and conjugation seemed to be equally important. The most important differences between the steady-state and single-dose groups were that in the steady-state group there was significantly more unchanged digitoxin, far less digoxin, and less hydroxylated metabolities than in the single-dose group. Caution is thus necessary when interpreting single-dose data for a drug that is used for maintenance.