To investigate whether atrial natriuretic factor regulates the growth of hepatocytes and to determine the receptor subtype involved in such modulation, we studied the effect of atrial natriuretic factor 103-126 and clearance receptor binding analogs of atrial natriuretic factor, (des-(Qfl6, S117, G1ls, L119, Glao) atrial natriuretic factor 102-121 and des-(C106J21) atrial natriuretic factor 104-126) on growth of human hepatoblastoma cells. Atrial natriuretic factor 103-126 and des-(Q116, S117, G118, L1lO, GlZo) atrial natriuretic factor 102-121 inhibited thymidine incorporation into human hepatoblastoma cells cultured in the presence of bovine serum albumin and epidermal growth factor but not in cells cultured in bovine serum albumin alone. Moreover, atrial natriuretic factor 103-126, des-(Q1l6, S117 G118 L119, GlZo) atrial natriuretic factor 102-121 and hes-(6106J21) atrial natriuretic factor 104-126, in a concentration-dependent manner, inhibited thymidine incorporation and cell proliferation. As monitored by the ability of des-(Q116, S117, G118, Ll'O, GlZo) atrial natriuretic factor 102-121 to displace 12SI-labeled atrial natriuretic factor, epidermal growth factor increased the expression of cell surface clearance receptors. Epidermal growth factor also transiently increased the cellular content of atrial natriuretic factor clearance receptor messenger RNA without altering the levels of guanylyl cyclase-linked atrial natriuretic factor receptor messenger RNA levels. Maximal increase in atrial natriuretic factor clearance receptor messenger RNA coincided with the maximal increase in des-(Q1l8, S"', G118, L119, GlZo) atrial natriuretic factor 102-12l-displaceable lz6Iatrial natriuretic factor binding sites. The ability of atrial natriuretic factor clearance receptor-binding ligands to inhibit thymidine incorporation and cell proliferation-coupled with the selective upregulation of clearance receptor expression by epidermal growth factor, which facilitates the growth modulatory actions of atriopeptins-indicates that the atrial natriuretic factor clearance receptors mediate the growth