Whole-body kinetics of 123I-labeled human insulin and five insulin analogues were investigated by scintigraphic studies in rats. The amino acid substitutions and the relative receptor affinities (RAs), determined by binding to HepG2 cells, were: GluB12, des-B30 insulin, RA 0.15%; AspB9, GluB27 insulin, RA 18%; AspB26 insulin, RA 80%; AspB18 insulin, RA 327%; and HisA8, HisB4, GluB10, HisB27 insulin, RA 687%. All analogues were compared with human insulin (RA 100%). The analogue with RA 0.15% showed a significantly slower disappearance in the heart window, no liver uptake, and the greatest kidney radioactivity, the latter probably caused by high plasma concentrations. The low-affinity analogue (RA 18%) reached a surprisingly high hepatic peak value, although significantly lower than insulin. Kidney radioactivity was higher than for insulin. The analogue with RA 80% showed liver and kidney radioactivities that were not significantly different from those of insulin. The two high-affinity analogues (RAs 327 and 687%) showed peak liver radioactivities not significantly different from insulin. However, liver radioactivity after the peaks declined significantly more slowly. Compared with insulin, the kidney radioactivity curves were not significantly different. We conclude that high-affinity insulin analogues will bind to any available receptor that, because of the large number of receptors in the periphery and the distribution of cardiac output, favors extrahepatic elimination. In contrast, low-affinity analogues bind to receptors several times before they are eliminated. This leads to recirculation and, thus, hepatic elimination due to the high receptor density there. It follows that hepatopreferential binding cannot be expected solely by use of an insulin analogue with a particular receptor affinity.