The intracellular concentrations of cyclic AMP, polyphosphoinosides and free Ca2+ were unaffected during receptor-mediated endocytosis of the neoglycoprotein beta-D-galactosyl-bovine serum albumin (D-Gal-BSA) by isolated hepatocytes. Elevation of either intracellular cyclic AMP by glucagon or inositol phosphates and Ca2+ by vasopressin were without effect on the binding and internalization of D-Gal-BSA. The normal response of this cell to glucagon- and vasopressin-mediated mobilization of these second messengers was not modified in the presence of saturating concentrations of D-Gal-BSA. Receptor-mediated endocytosis of diferric transferrin (Fe3+-TRF) by both hepatocytes and HL60 cells was also shown to be independent of second messengers, although the unequivocal expression of the transferrin receptor by hepatocytes could not be satisfactorily demonstrated. The results of the present study are at variance with a suggested regulatory role for second messengers in receptor-mediated endocytosis of serum-derived ligands such as asialoglycoproteins and Fe3+-TRF. Receptor phosphorylation by protein kinase C in particular has been proposed to regulate the distribution and recycling of these receptors in actively endocytosing cells. We would suggest that if receptor phosphorylation has a regulatory role during endocytosis, it is likely to be mediated by a second-messenger-independent protein kinase analogous to casein kinase II. An alternative interpretation is that phosphorylation has no physiological significance and receptor-mediated endocytosis is a constitutive event coupled to membrane turnover.