Two leucines (Leu986 and Leu987) have recently been shown to take part in the control of human insulin receptor (HIR) internalization (Renfrew-Haft, C., Klausner, R. D., and Taylor, S. I. (1994) J. Biol. Chem. 269, 26286-26294). The aim of the present study was to further investigate the exact mechanism of this control process. Constitutive and insulin-induced HIR internalizations were studied biochemically and morphologically in NIH 3T3 cells overexpressing either a double alanine (amino acid residues 986-987) mutant HIR (HIR AA1) or HIR truncated at either amino acid residue 981 (HIR Delta981) or 1000 (HIR Delta1000). Data collected indicate that: (a) the three mutant HIR show a reduced association with microvilli as compared with HIR wild-type; (b) the two receptors containing the dileucine motif (HIR WT and HIR Delta1000) show the highest propensity to associate with clathrin-coated pits, independently of kinase activation; (c) the two receptors lacking the dileucine motif but containing two tyrosine-based motifs, previously described as participating in clathrin-coated pit segregation, associate with these surface domains with a lower affinity than the two others, (d) in the presence of the kinase domain, an unmasking of the tyrosine-based motifs mediated by kinase activation is required. These results indicate that the dileucine motif is not sufficient by itself, but participates in anchoring HIR on microvilli and that another sequence, located downstream from position 1000 is crucial for this event. This dileucine motif also plays a role in HIR segregation in clathrin-coated pits. This latter function is additive with that of the tyrosine-based motifs but the role of the dileucine motif predominates. Eventually, the clathrin-coated pit anchoring function of the dileucine motif is independent of receptor kinase activation in contrast to the tyrosine-based motifs.