Abstract The subcellular localization of metformin was studied in livers of 18 h fasted rats treated orally with [14C]metformin 20 μCi kg−1, 50 mg kg−1. Sequential determination of 14C radioactivity showed that maximum concentrations of metformin in plasma (about 15 μmol L−1) and liver (about 50 μmol kg−1) were achieved at 30–60 min and approximately half-maximal concentrations were achieved at 4 h. At 30 min and 4 h after administration of [14C]metformin, livers were removed and homogenized. Nuclear, mitochondrial and lysosomal, mixed membrane, and cytosolic fractions were separated by ultra-centrifugation. Distribution of 14C was similar at both time points, being greatest in the cytosolic fraction (78% of total radioactivity, and 60–69% of relative specific activity). Small amounts of 14C were associated with the other fractions. The total radioactivity and relative specific activity were respectively 2–3% and 7–8% in the nuclear fraction, 9–10% and 7–10%) in the mitochondrial and lysosomal fraction, and 8–9% and 13–14% in the mixed membrane fraction. The higher concentration of metformin in liver compared with plasma suggests that metformin enters hepatocytes via a specific mechanism, and is distributed mainly within the cytosol.