Biological activity and interference with insulin receptor complex fate of two modified sequences of insulin B21-B26, beta-Ala-Arg-Gly-Phe-Phe-Tyr-NH2 (DP-432) and beta-Ala-Arg-Pro-Phe-Phe-Tyr-NH2 (DP-640), were studied in cultured 18-day-old fetal rat hepatocytes known to respond to insulin by an acute stimulation of glycogenesis. The two derivatives stimulated [14C]glucose incorporation into glycogen in the absence of insulin independently of the deprivation of serum in the medium. The maximal effect of 3 mM DP-640 after 2 h, more pronounced than with 3 mM DP-432, was of the same order as that obtained with 10 nM insulin alone (stimulation index: 4.7 +/- 0.7, 2.5 +/- 0.2 and 3.6 +/- 0.9, n = 4, with DP-640, DP-432 and insulin, respectively) whereas insulin B-chain decreased glycogen labeling. Simultaneous addition of derivatives and insulin at maximal concentrations produced nearly additive effects. DP-640, as well as DP-432, increased the amount of [125I](A14) or (B26) human insulin associated with cells at 37 degrees C and inhibited intracellular insulin degradation with differences depending on the kind of insulin isomer and derivative, while the rapid insulin receptor cycle was not affected. Thus, the two derivatives specifically modified the cellular processing of insulin in cultured fetal hepatocytes, and exerted an insulin-like effect on glycogenesis clearly enhanced through modification of DP-432 by substitution of glycine for proline (DP-640).