Asialoglycoprotein receptors (ASGP-Rs) in permeable rat hepatocytes can be inactivated in the absence of ligand. This cytosol-independent effect is relatively slow (t1/2 approximately 12 min) and is temperature and ATP dependent. Here we show that in the absence of cytosol, the addition of palmitoyl-CoA (Pal-CoA) rapidly (t1/2 < 0.4 min) and quantitatively reactivates the inactivated receptors. Receptor reactivation was half-maximal at approximately 10-12 microM free Pal-CoA at 37 degrees C. Although substantially higher total concentrations were used, much of the added Pal-CoA was cell associated and not free. The effects of Pal-CoA were eliminated by bovine serum albumin at concentrations sufficient to bind all free monomeric fatty acyl-CoA, suggesting that micellar effects are not responsible for the ability to reactivate ASGP-Rs. Also, palmitoyl-carnitine did not substitute for Pal-CoA. The initial ASGP-R inactivation is not affected by treating cells with N-ethylmaleimide or by a KCl wash but is inhibited by sodium orthovanadate or high Ca2+ levels. Myristoyl-CoA (C14) was also able to reactivate inactive ASGP-Rs about as well as Pal-CoA. Fatty acyl-CoAs with chain lengths of C12 (lauroyl) or C18 (steroyl) were < 50% as active. The ligand binding activity of these receptors can subsequently be modulated within minutes by the further addition of ATP or Pal-CoA to achieve additional rounds of ASGP-R inactivation or reactivation, respectively. These in vitro data demonstrate the occurrence of a novel asialoglycoprotein receptor inactivation-reactivation cycle that could regulate receptor activity during endocytosis and receptor recycling.