We have investigated the functional relationships between insulin receptor (IR) trafficking, mitogenic signaling and mRNA expression in rat liver and primary hepatocytes. The low-K(d) insulin analogues [His(A8),His(B4), Glu(B10),His(B27)]-human insulin (-HI) (the H2-analogue), [Asp(B10)]HI and [Glu(A13),Glu(B10)]HI, were studied in liver parenchymal cells and compared with wild-type HI and epidermal growth factor (EGF), a mitogenic inducer. The extent and duration of IR endocytosis were markedly increased in response to the H2-analogue and [Asp(B10)]HI compared to wild-type HI, but similar to HI after [Glu(A13),Glu(B10)]HI administration. Importantly, the insulin analogues induced a higher and more prolonged tyrosine phosphorylation of the IR-beta subunit in endosomes compared to authentic HI. A low cell-free endosome-lysosome transfer of the internalized IR was only observed in response to HI and H2-analogue injection. Concomitant with the low endosome-lysosome transfer of the intact IR-beta subunit, 47 and 50 kDa fragments of the IR-beta subunit accumulated in lysosomal fractions. Neither HI nor the insulin analogues promoted the endosomal recruitment and tyrosine phosphorylation of Shc, whereas EGF accessed the Shc signaling pathway. Moreover, EGF induced a fast and prolonged activation of Raf-1 and MAP-kinase pathways whereas HI and insulin analogues displayed a moderate and transient effect. Finally, treatment of primary rat hepatocytes with HI and the protease-resistant H2-analogue did not affect the total level and relative expression of isotype A and B of IR mRNA regardless of time of exposure. These results suggest a lack of relationship between IR trafficking, endosomal tyrosine phosphorylation and mitogenic signaling in rat liver in vivo.