HOE 901 is a new biosynthetic long-acting human insulin analog (GLY[A21]ARG[B31]ARG[B32]). We compared HOE 901 with normal human insulin and the insulin analog Asp(B10), which is known to have increased mitogenic activity at least partially mediated through the insulin receptor. We have analyzed receptor binding, insulin-induced receptor autophosphorylation and phosphorylation of receptor substrates in rat-1 fibroblasts overexpressing human insulin receptor isoform A (HIR A) or B (HIR B). In HIR A expressing cells, insulin and its analogs showed no significant differences in receptor association while clearly different dissociation kinetics were observed. In HIR B expressing cells, no significant differences in association and dissociation kinetics were observed. All insulins induced rapid autophosphorylation of the insulin receptor reaching a maximum after 10 min of stimulation. Asp(B10)insulin induced a prolonged phosphorylation state (over 60 minutes) of the 95 kDa receptor beta-subunit and of the substrates IRS-1/IRS-2 and Shc in contrast to normal human insulin and to HOE 901. In addition, we observed an increased and prolonged tyrosine phosphorylation of an unidentified protein with Asp(B10)insulin at about 60 kDa. Insulin-dependent dephosphorylation of the focal adhesion kinase (p125FAK) was equally induced by all these ligands. With respect to [3H]thymidine incorporation into DNA, HOE 901 had similar effects as normal human insulin, while Asp(B10)insulin showed increased [3H]thymidine incorporation. In summary, the data show that the increased mitogenic activity of Asp(B10)insulin may be explained with a prolonged kinetics of tyrosine phosphorylation of the insulin receptor and of insulin signalling elements together with the preferential phosphorylation of an yet unidentified 60 kDa protein. HOE 901 behaves with respect to insulin receptor binding, receptor autophosphorylation, phosphorylation of signalling elements and promotion of mitogenesis like regular human insulin.